Identification of a Novel PP2A Regulator, WNK1, as Critical For Uterine Function

1 Reproductive and Developmental Biology Laboratory, National Institute of Environmental 5 Health Sciences, Durham, NC, 27709, USA 6 2 Integrative Bioinformatics Support Group, National Institute of Environmental Health Sciences, 7 Durham, NC, 27709, USA 8 3 Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa, IA, 9 52242, USA 10 4 Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel 11 Hill, NC, 27599, USA 12 5 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 13 77030, USA 14

Here, we employed a mouse model with conditional WNK1 ablation from the female 23 reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine that WNK1 is needed for proliferation, migration and differentiation 3 . Collectively, these findings 48 indicate a previously unrecognized function of WNK1 in the female reproductive tract and led us 49 to hypothesize that WNK1 is a mediator of uterine function. 50 51 WNK1 belongs to a family of serine/threonine protein kinases 4, 5 , with its name derived from the 52 unusual placement of the catalytic lysine in subdomain I 6 . To date, Wnk1's function is the most 53 extensively explored in the kidney and the nervous system due to the link between its mutation 54 and familial hypertension and autonomic neuropathy 7-9 . In the renal system, WNK1 controls ion 55 homeostasis through diverse mechanisms including activation of the SGK1/epithelial sodium 56 channel pathway 10 , regulating the potassium channel Kir1.1 cell surface localization 11 , as well as 57 controlling the activity of Na-K-Cl cotransporter through phosphorylating OSR1(OXSR1) and 58 SPAK 12, 13 . Interestingly, WNK1's regulatory function on OSR1 is critical for cardiovascular 59 development, thereby contributing to embryonic lethality when WNK1 is ablated from the 60 endothelium 14, 15 . These findings suggest that although WNK1 exhibits organ-specific 61 of gland reorientation surrounding the embryo 19,20 . Examination of uterine cross sections from 100 older mice (26 and 50 weeks) further demonstrated invasion of glands into the myometrium, 101 suggesting that WNK1 ablation caused adenomyosis (Fig. 2.B). This was supported by the 102 elevated expression of Moesin (Msn) in the Wnk1 d/d uteri, a biomarker for adenomyosis in humans 103

Uterine WNK1 ablation compromised fertility in mice through impaired implantation 117
A six month breeding trial was conducted to determine the impact of WNK1 ablation on female 118 fertility. Of the 8 control mice, 7 were able to complete the breeding trial, with 1 found dead 119 midtrial. Necropsy showed neither pregnancy nor abnormality associated with the reproductive 120 tract in this one mouse, indicating the cause of death was not related to abnormal uterine function. 121 The 7 mice produced 31 litters totaling 245 pups, which was equivalent to 4.4 litters and 35 pups 122 per mouse during the 6 months ( Fig. 3.A and B). In contrast, only 4 of the 8 Wnk1 d/d mice initiated 123 in the trial were able to complete the trial. This was due to 4 females succumbing to complications 124 regulation of this signaling pathway. Moreover, the underlying stromal cells also exhibited reduced 174 nuclear FOXO1, suggesting that the cellular impact of WNK1 on FOXO1 localization may be 175 similar in both the epithelial and stromal compartments. Our findings illustrated that several crucial 176 implantation-associated molecular events were deregulated in the Wnk1 d/d mice. 177 178

Wnk1 d/d mice 180
Interestingly, of the 29.4% mated Wnk1 d/d mice that were able to permit embryo implantation on 181 time (GD 4.5), the number of implantation sites were similar to their Wnk1 f/f control littermates 182 ( Fig. 4.A). However, the number of implantation sites present on GD 5.5 was significantly lower 183 in the Wnk1 d/d mice (Fig. 4.B). This finding indicated that the delay in implantation is associated 184 with reduced number of implantation sites. Additionally, spacing between the implantation sites in 185 the Wnk1 d/d mice were irregular whereas the implantation sites observed in the Wnk1 f/f mice were 186 evenly distributed (Fig. 4.C). Interestingly, for the Wnk1 d/d mice that were able to implant promptly, 187 implantation spacing was more evenly distributed ( gestation sac size (Fig. 4.F and G) and decreased embryo size at both GD 8.5 and GD 10.5 (Fig.  198 4.F and H). By GD 12.5, embryo resorption was frequently observed in the Wnk1 d/d mice (Fig. 199 4.F, bottom panel). Collectively, these findings demonstrate that uterine WNK1 ablation led to 200 abnormal implantation and negatively impacted embryo development, resulting in the 201 compromised pregnancy outcome and subfertility.. To fully characterize the molecular mechanisms underlying the loss of WNK1 induced-206 implantation defect, we next examined global gene expression profile by RNA sequencing (RNA-207 seq) in the uterus during receptivity. To ensure that the analysis was conducted only on the 208 maternal uterine tissues and not the embryos, we used vasectomized wild-type males to induce 209 pseudopregnancy in the Wnk1 f/f and Wnk1 d/d mice, which was confirmed by serum progesterone 210 levels on pseudopregnancy day (PPD) 4.5 (Table S1). In total, there were 14,423 and 14,337 211 genes expressed in the Wnk1 f/f and Wnk1 d/d uterus, respectively; of which 14,024 were expressed 212 in both. The transcriptomes were subjected to principle component analysis (PCA) as a measure 213 of quality control, which segregated according to genotype indicating that the samples were well-214 characterized by genotype (Fig. S3). Using a defining threshold of q-value under 0.05 for 215 significance and fold change (FC) over 1.5 as differential expression, we identified 1,727 216 significantly and differentially expressed genes (DEGs) in the Wnk1 d/d uterus during receptivity 217 (Table S2). We then conducted detailed analyses to characterize the molecular alterations 218 associated with uterine WNK1 ablation using the DAVID Bioinformatic Database and Ingenuity 219 Pathway Analysis (IPA). The top biological processes associated with the DEGs were adhesion, 220 cell movement and locomotion, inflammation and blood vessel development (Table S3). Many 221 important molecular functions associated with implantation were also deregulated in the Wnk1 d/d 222 uteri, such as cell proliferation and apoptosis, Notch signaling, cell differentiation, epithelial to 223 mesenchymal transition (EMT), cytokine production, and response to estrogen. Prediction of 224 upstream regulator activity further showed altered activity for many important receptivity 225 mediators, including the suppression of JAG, HEY2, PTEN and SERPINE1 (Fig. 5.A). On the 226 other hand, TGFB1, ERBB2, AKT, estrogen, ERK, MUC1 and KLF5 were predicted to show 227 increased activity (Fig. 5.A, for the complete list see Table S4). 228

229
We have earlier shown that there was a decrease in nuclear FOXO1 during the window of 230 implantation in the Wnk1 d/d mice ( Fig. 3.I). Since FOXO1 is a transcription factor that is critical for 231 implantation, its nuclear exclusion would likely results in its reduced activity in transcribing genes. 232 Therefore, we compared the DEGs to known FOXO1-controlled endometrial genes 16 , and found 233 that of the 631 FOXO1-regulated endometrial genes, 313 showed altered expression in Wnk1 d/d 234 uterus (Fig. 5.B). Detailed comparison further illustrated that 90% of these common genes were 235 deregulated in the same direction under WNK1 and FOXO1 deficient conditions ( Fig. 5.C). These 236 findings suggest that the impaired implantation observed in the Wnk1 d/d mice was mediated 237 partially through insufficient FOXO1 signaling during receptivity.  Fig. S4.B). Furthermore, elevated AKT phosphorylation on GD 250 4.5 was confirmed in both the epithelium and the stroma (Fig. 5.F). Indeed, we found that in the 251 control mice, phosphorylation of AKT was actively suppressed as the mice transitioned into the 252 receptive phase from GD 3.5 to PPD 4.5, however, the Wnk1 d/d mice maintained high AKT 253 phosphorylation both prior to and during receptivity (Fig. 5.G). As AKT directly phosphorylates 254 FOXO1 leading to its nuclear exclusion 27, 28 , and decreased nuclear FOXO1 was observed in the 255 Having demonstrated that the loss of WNK1 led to increased phosphorylation of AKT and FOXO1 264 in mouse uteri, we next examined whether this regulatory axis was similarly maintained in human 265 endometrial HEC1A (epithelial) and THESC (stromal) cells. Using small interfering RNA against 266 WNK1 (siWNK1), WNK1 protein expression was inhibited, which robustly induced AKT and 267 FOXO1 phosphorylation in both cell lines (Fig. 6.A). In order to test whether AKT facilitated 268 FOXO1 nuclear exclusion downstream of WNK1, we next treated these cells with an AKT 269 inhibitor, GDC0941, and examined whether it could rescue WNK1 ablation-induced 270 phosphorylation and nuclear exclusion of FOXO1. FOXO1 localization clearly decreased in the 271 nucleus of both cells after transfection with siWNK1 ( Fig. 6.B, panels 1 VS 2, and 4 VS 5). 272 However, when the siWNK1 transfected cells were treated with GDC0941, nuclear FOXO1 was 273 readily restored ( Fig. 6.B, panels 3 and 6). This suggested that WNK1 inhibition-induced nuclear 274 exclusion of FOXO1 is mediated through AKT. This is further supported by the findings that AKT 275 inhibition rescued WNK1 knock-down induced FOXO1 phosphorylation ( Fig. 6.C). Interestingly, 276 GDC0941 treatment reduced the phosphorylation of AKT and FOXO1 to a level that is lower than 277 seen in the siCTRL transfected, untreated cells (considered basal level). As GDC0941 inhibits 278 AKT through its upstream regulator PI3K 29 , it is likely that PI3K lies upstream of WNK1 in 279 regulating AKT. Indeed, none of the PI3K family members were impacted by WNK1 inhibition, 280 including p110-α, p110-β, p110-γ, Tyr458 phosphorylated p85 and Tyr199 phosphorylated p55 281  Table  286 S4). PTEN and PPP2CA are both phosphatases that regulate AKT phosphorylation, and both 287 displayed repressed activities in the Wnk1 d/d mice during receptivity (Z-scores of -2.079 and -288 1.195, respectively, Table S4). Sirolimus, on the other hand, is a drug targeting the kinase mTOR, 289 which was strongly inhibited (Z-score of -2.95, Table S4). We found that mTOR phosphorylation 290 and PP2A subunits A and C were altered in the Wnk1 d/d mice, while PTEN level was not 291 significantly different (Fig. 6.E). This finding suggested that increased AKT phosphorylation in the 292 Wnk1 d/d mice may be mediated through elevated mTOR or repressed PP2A activity. As mTOR is 293 both a regulator and a substrate of AKT 30, 31 , we examined whether WNK1 ablation-induced AKT 294 phosphorylation is mediated through mTOR. We inhibited mTOR activity using rapamycin and 295 examined AKT/FOXO1 phosphorylation as well as FOXO1 localization as a readout of AKT 296 activity. As shown in Fig. S5.A, rapamycin treatment did not reverse the nuclear exclusion of 297 FOXO1 induced by WNK1 inhibition. Additionally, AKT and FOXO1 phosphorylation was not 298 rescued by rapamycin treatment (Fig. S5.B). Similar results were observed in HEC1A cells where 299 WNK1 and mTOR double knock-down failed to rescue AKT and FOXO1 phosphorylation (Fig.  300 S5.C). Thus, mTOR is likely not the WNK1 mediator controlling AKT activity, and its elevated 301 phosphorylation is a result of elevated AKT activity, rather than its cause. 302

WNK1 regulates AKT phosphorylation through direct interaction with PPP2R1A 304
WNK1 ablation led to impaired implantation arising from dysregulated AKT-FOXO1 signaling with 305 a concomitant repression of PP2A activity and reduced PP2A subunits A and C expression. We 306 explored the possible regulatory link between WNK1 and PP2A/AKT using a non-biased WNK1 307 immunoprecipitation-mass spectrometry (IP-MS) approach to identify WNK1 binding partners. 308 Successful WNK1 IP was confirmed by examining the lysate for WNK1 expression after 309 immunoprecipition using a a rabbit IgG (negative control) or WNK1 targeting antibody from 310 HEC1A cells (Fig. S6), and the peptides identified by mass-spectrometry are listed in Table S5. 311 Amongst those were peptides belonging to WNK1 itself, as well as a known WNK1 substrate, 312 oxidative stress responsive kinase 1 (OXSR1/OSR1) 12 , confirming the validity of the pull-down 313 results (Table S5).  , Table S4), we postulated that these observations were associated with the direct 323 interaction of WNK1 and PPP2R1A, the alpha isoform of the scaffold subunit A of PP2A. In order 324 to confirm the interaction of WNK1 and PPP2R1A, a YFP-tagged WNK1 (c4161, Fig. S7) was 325 expressed in HEC1A cells, then immunoprecipitated using a YFP nanobody, followed by detection 326 for PPP2R1A in the pulldown. We first confirmed that c4161 transfection induced exogenous 327 WNK1 expression when compared to the control cells transfected with YFP only expressing 328 construct (cYFP, Fig. 7.A). WNK1 was subsequently detected in the lysate immunoprecipitated 329 for YFP ( Fig. 7.B, upper panel), which co-immunoprecipitated with PPP2R1A ( Fig. 7 Having confirmed the interaction of WNK1 and PPP2R1A, we next explored the biological 333 implications of this interaction. The PP2A phosphatase complex is comprised of the scaffold 334 subunit A with 2 isoforms, the regulatory subunit B with 13 isoforms and the enzymatic subunit C 335 with 2 isoforms. As shown earlier, uterine WNK1 ablation led to decreased protein levels of 336 subunits A and C ( Fig. 6.E), yet RNA-seq showed no alteration in transcription of the 4 genes 337 encoding these 2 subunits in the Wnk1 d/d mice. It has been reported that the stability of the PP2A 338 subunits depends on their association with each other 32 . Hence, reduced subunit levels could be 339 an indication that the complexing capacity of the subunits were reduced in the absence of WNK1, 340 leading to their degradation. We therefore postulated that the WNK1-PPP2R1A interaction is 341 necessary for the association of the PP2A subunits. To test this idea, we examined the levels of 342 PPP2R1A, total PP2A subunit A and total PP2A subunit C in WNK1 knock-down HEC1A cells, 343 and accordingly found their reduced levels upon WNK1 inhibition (Fig. 7.C). Lastly, to validate 344 that PP2A mediates AKT/FOXO1 signaling, we inhibited PPP2R1A expression in HEC1A cells 345 using siRNA, and examined the components of the PP2A-AKT-FOXO1 signaling axis. As 346 expected, PPP2R1A knock-down caused a reduction in total subunits A and C of PP2A (Fig. 7.D). 347 Interestingly, AKT phosphorylation was selectively induced on threonine 308, but not serine 473 348 after PPP2R1A knock-down ( Fig. 7.D). This nonetheless, translated to elevated FOXO1 349 phosphorylation, indicating that loss of PP2A activity-induced AKT phosphorylation on this residue 350 alone is sufficient to alter FOXO1 phosphorylation (Fig. 7.D). These findings confirmed that in 351 endometrial cells, WNK1 controls FOXO1 phosphorylation through directly interacting with 352 PPP2R1A, which leads to stabilization of the PP2A complex and induces AKT dephosphorylation 353 Reproductive biology has relied profoundly on transcriptomic analyses to identify novel players 360 that may serve crucial functions in the regulation of fertility. While this approach has uncovered 361 many key components in the reproductive tract, it is unable to detect alterations at the proteomic 362 level, such as post translational modifications (PTMs). In many cases, the PTMs control protein 363 activity and stability, and hence are the actual determinants of functional output. Using a 364 proteomic approach, we identified WNK1 as a potential regulator of uterine biology with previously 365 unreported reproductive functions. In this study, we examined the in vivo function of WNK1 using 366 a whole-uterus WNK1 knock-out mouse model. We demonstrate that loss of WNK1 led to 367 hyperplasia, adenomyosis and impaired implantation, which could all contribute to compromised 368 fertility. Importantly, we demonstrate for the first time that WNK1 is a direct regulator of the PP2A-369 AKT-FOXO1 signaling pathway. As FOXO1 is critical for embryo implantation in mice 16 , and the 370 subfertile phenptype of Wnk1 d/d mice is associated with implantation impairments, it is likely that 371 aberrant FOXO1 localization is at least partially responsible for the subfertility. It is worth noting 372 that the Wnk1 d/d mice did not fully recapitulate the uterine FOXO1 knock-out phenotype, possibly 373 due to decreased FOXO1 signaling, rather than complete inhibition. This finding suggests that WNK1 ablation-induced adenomyosis may share molecular similarities 428 to this disease in humans. We propose that the uterine WNK1 ablation mouse model could be 429 employed to study this disease. peroxidase was performed by treating the sections with 3% hydrogen peroxide diluted in distilled 535 water for 10 minutes at room temperature. Tissues were blocked in 5% normal donkey serum 536 (NDS) for 60 minutes at room temperature, prior to overnight incubation with the primary antibody 537 at 4 O C. The slides were washed twice in PBS for a total of 10 minutes at room temperature and 538 secondary antibody diluted in 1% w/v bovine serum albumin (BSA) prepared in PBS was applied. 539 The ABC reagent was applied to tissue according to the manufacturer's instructions (Vector Labs 540 ABC PK-6100, Vector Laboratories). Signals were developed using the Vector Labs DAB 541 ImmPACT Staining Kit (Vector Labs SK-4105, Vector Laboratories). Finally, the tissues sections 542 were counterstained with hematoxylin and dehydrated through increasing ethanol concentration, 543 followed by Citrisolv incubation and coverslipping. For immunofluorescence, tissue sections were 544 subjected to antigen retrieval as described above. Tissues were blocked in 0.4% v/v Triton X-100, 545 1% BSA and 5% NDS for 30 minutes at room temperature followed by overnight incubation in

RNA extraction and cDNA conversion 554
The frozen tissues were disrupted in TRIzol reagent (Thermo Fisher) by bead milling, followed by 555 2 aqueous phase separations using 1-Bromo-3-chloropane and chloroform. Pure ethanol was 556 added to the aqueous layer, and the RNA was extracted using the Qiagen RNEasy RNA mini 557 prep kit columns as per manufacturer's instructions (Qiagen, Valencia, CA). Resulting RNA 558 concentration and quality was determined using the NanoDrop ND-1000. cDNA was generated 559

Human phospho-kinase antibody array 588
Site specific phosphorylation levels of 43 kinases were measured using the Human Phospho-589 Kinase Array Kit (catalog no. ARY 003B, R&D Systems) according to the manufacturer's 590 instructions with the experimental design as described below. Pseudopregnancy was induced in 591 the Wnk1 f/f and Wnk1 d/d mice as previously described, and mice were euthanized on PPD 4.5. 592 Uterine tissues were frozen at -80 O C until ready to proceed. Lysate were extracted independently 593 from 6 mice per group by bead milling in the lysis buffer provided within the kit, and protein 594 concentrations were determined using the BCA Protein Assay Kit (catalog no. 23225, Pierce). 595 Equal amounts from each mouse were pooled in each group (to a total of 900 µg), and the 596 remaining steps followed the standard protocol of the kit. Signal intensity was quantified by 597 ImageJ 56 . 598 599

Protein extraction from uterine tissues and protein expression analysis 600
Tissues were homogenized in RIPA Lysis and Extraction Buffer (Thermo Fisher) supplemented 601 with protease inhibitor cocktail (cOmplete Mini, EDTA-free, catalog no. 11836170001, Roche 602 Diagnostics) and phosphatase inhibitor cocktail (phosSTOP, catalog no. 4906837001, Roche 603 Diagnostics), followed by centrifugation at 10,000 X G for 10 minutes at 4 O C, and the supernatant 604 was moved into fresh eppendorf. Protein concentrations were measured using the BCA Protein 605 Assay Kit (Pierce). Heat denatured protein samples were resolved using 7.5%, 10% or gradient 606 4-20% Criterion Tris-HCl precast gels (Bio-Rad), followed by transferring using the Trans-Blot HEC1A cells were grown to 70% confluency, followed by collection using trypsin. Cells were 681 washed 2 X in cold PBS, followed by resuspension in cell lysis buffer (50 mM Tris-HCl pH 7.5, 682 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, with protease and 683 phosphatase inhibitors added fresh to 1 X). Cells were incubated on ice for 10 minutes, followed 684 by sonication on medium power (3 X 5 seconds). Lysate was centrifuged at 13,000 X G for 10 685 minutes at 4 O C. WNK1 targeting antibody was added at 1:100 to the supernatant, and incubated 686 with rotation at 4 O C overnight. Prewashed beads (50% protein A and 50% protein G, catalog 687 numbers 10002D and 10004D, respectively, Thermo Fisher) were added to the immunocomplex 688 and incubated for 30 minutes at room temperature with rotation. Beads were pelleted using a 689 magnetic separation rack, followed by 3 washes in lysis buffer. Beads were heated to 100 O C with 690 3 X SDS buffer (150 mM Tris-HCl pH 6.8, 6% SDS, 0.3% BPB, 30% glycerol, 3% B-691 mercaptoethanol) for 5 minutes, before electrophoresis through a 7.5% Criterion Tris-HCl precast 692 gel (Bio-Rad). Gel regions were excised from the SDS-PAGE gel and minced, and digests were 693 performed with a ProGest robotic digester (Genomic Solutions) where the gel pieces were 694 destained by incubation in 25 mM ammonium bicarbonate with 50% acetonitrile (v/v) twice for a 695 total of 30 minutes. The gel pieces were dehydrated in acetonitrile, followed by drying under a 696 nitrogen stream, and further incubated with 250 ng trypsin (Promega) for 8 hours at 37 O C. The 697 digests were collected, and peptides were re-extracted three times. The extractions were pooled 698 for each sample, lyophilized and resuspended in 20 µL 0.1% formic acid. The protein digests were 699 analyzed by LC/MS on a Q Exactive Plus mass spectrometer (Thermo Fisher) interfaced with a 700 nanoAcquity UPLC system (Waters Corporation), and equipped with a 75 µm x 150 mm BEH 701 dC18 column (1.8 µm particle, Waters Corporation) and a C18 trapping column (18 µm x 20 mm) 702 with a 5 µm particle size at a flow rate of 400 nL/min. The trapping column was positioned in-line 703 of the analytical column and upstream of a micro-tee union which was used both as a vent for 704 trapping and as a liquid junction. Trapping was performed using the initial solvent composition. A 705 volumn of 5 µL of digested sample was injected into the column, and peptides were eluted by 706 using a linear gradient from 99% solvent A (0.1% formic acid in water (v/v)) and 1% solvent B 707 (0.1%formic acid in acetonitrile (v/v)), to 40% solvent B over 60 minutes. For the mass 708 spectrometry, a data dependent acquisition method was employed with an exclusion time of 15 709 seconds and an exclusion of +1 charge states. The mass spectrometer was equipped with a 710 NanoFlex source and was used in the positive ion mode. Instrument parameters were as follows: 711 sheath gas, 0; auxiliary gas, 0; sweep gas, 0; spray voltage, 2.7 kV; capillary temperature, 275 O C; 712 S-lens, 60; scan range (m/z) of 200 to 2000; 2 m/z isolation window; resolution: 70,000; automated 713 gain control (AGC), 2 X 10 5 ions; and a maximum IT of 200 ms. Mass calibration was performed 714 before data acquisition using the Pierce LTQ Velos Positive Ion Calibration mixture (Thermo 715 Fisher). Peak lists were generated from the LC/MS data using Mascot Distiller (Matrix Science) 716 and the resulting peak lists were searched using the Spectrum Mill software package (Agilent) 717 against the SwissProt database. Searches were performed using trypsin specificity and allowed 718 for one missed cleavage and variable methionine oxidation. Mass tolerance were 20 ppm for MS 719 scans and 50 ppm for MSMS scans. 720 721

Generation of mammalian YFP-WNK1 expression constructs 722
The coding region of the WNK1 sequence (NM_014823.3) with attL sites and N-terminal TEV 723 cleavage site was synthesized by GeneWiz Inc. and cloned into pUC57 (Kanamycin) plasmid. 724 Gateway Cloning using LR Clonase II mix (Thermo Fisher) was used to transfer the WNK1 725 sequence into the Vivid Colors pcDNA6.2/N-YFP vectors (Thermo Fisher), which created the 726 mammalian expression vectors with YFP fused to the N-terminal end of WNK1 (Fig. S7, c4161). 727 728

Co-Immunoprecipitation 729
HEC1A cells were transfected with cYFP or c4161 for 48 hours, followed by trypsinization, 3 730 washes and resuspension in lysis buffer (50 mM Tris pH8.0, 400 mM NaCl, 0.1% NP-40 and 0.5 731 mM DTT, with protease and phosphatase inhibitors freshly added to 1 X). The lysate was 732 incubated at 4 O C with rotation for 30 minutes. Lysates were centrifuged at 21,100 X G for 10 733 minutes at 4 O C, and the supernatant was added to 1.5 volumes of 25% glycerol, followed by 734 centrifugation at 21,100 X G for 10 minutes at 4 O C. Anti-GFP resin slurry was added to the 735 supernatant and nutated for 1 hour at 4 O C. Beads were centrifuged at 1,000 X G for 5 minutes,