(A) shRNA-mediated synectin knockdown in hHSC decreases both PDGFR-α and PDGFR-β protein levels as depicted by Western blot. Densitometry located in the graph below, n = 5. (B) Control or synectin-knockdown hHSCs were treated with the proteosomal inhibitor MG132 (25 μM) for 4 hours; however, in PDGFR-α or -β, protein levels were not increased by proteosomal inhibitor MG132 (n = 3) in hHSC with synectin knockdown. (C and D) In contrast, PDGFR-α levels were increased by inhibition of autophagy by bafilomycin (10 μM, overnight) and 3-MA (1 μM, overnight) in both control and synectin-knockdown hHSC, n = 3. (E) Inhibition of autophagy by ATG5 siRNA increased PDGFR-α protein levels in hHSCs, n = 3. (F) LX-2 cells were transfected with a LacZ control plasmid, a plasmid encoding WT NRP-1 (NRP-1-WT), or a plasmid encoding a NRP-1 mutant lacking the SEA domain (NRP-1-ΔSEA), and PDGFR-α protein levels were determined by Western blot. Overexpression of WT NRP-1 enhanced PDGFR-α expression; however, the NRP-1 mutant resulted in decreased PDGFR-α protein levels, n = 3. (G) siRNA-mediated NRP-1 knockdown in hHSC showed reduction in PDGFR-α protein levels without affecting PDGFR-β protein levels as shown by Western blot, n = 7. (H) Bafilomycin (10 μM) treatment increased PDGFR-α protein levels following siRNA-mediated NRP-1 knockdown as observed by Western blot, n = 3. (I) hHSCs were transfected with NRP-1 siRNA or a control, followed by treatment with MG132 (25 μM). PDGFR-β protein levels were not increased by MG132 treatment in NRP-1–knockdown cells, n = 3. Samples were run on the same gel but were noncontiguous in B–E and I. Data are expressed as mean ± SEM. Densitometry for all experiments was analyzed by ImageJ and are depicted below their respective blots. One-way ANOVA with Bonferroni’s multiple comparison test was used to analyze groups for statistical significance (*P < 0.05, **P < 0.001, ***P < 0.0001).