(A) Western blots of lysates from nontransfected HEK293T cells and HEK293T cells transfected with a vector encoding HA-tagged wild-type (HA CALM-WT) or mutant CALM (HA CALM-N98S). Cells were harvested before and after 6-hour, 12-hour, 24-hour, or 36-hour cycloheximide (CHX; 200 mg/mL) treatment. Control cells (labeled -) were harvested 36 hours after addition of the solvent (ethanol) only. Lysates were probed with an antibody recognizing the C-terminus of CALM or the HA-tag fused to the N-terminus of CALM. Samples were normalized to GAPDH protein expression. (B) Changes in normalized intensities of the endogenous CALM protein and HA-CALM protein bands expressed as percentages of their corresponding time 0 values and plotted as a function of CHX exposure time. Shown are dot plots with lines representing medians and interquartile ranges (N = 7–14 biological replicates per genotype). Each biological replicate is the average of 2 technical replicates. Numbers next to brackets denote P values by a repeated measures model with independent variables of plasmid, hour, and interaction of plasmid and hour. (C) Western blots of lysates from HEK293T cells transfected with HA CALM-WT, HA CALM-N98S, or control (labeled Empty) vectors. Lysates from cells following 24-hour exposure to MG132 (10 mmol/L) or lactacystin (10 mmol/L) were probed with the same anti-CALM or anti-HA antibodies as in A. Signal intensities were normalized to GAPDH protein expression. (D) Normalized intensities of HA-CALM protein bands expressed as fold-changes of normalized intensities in DMSO-treated cells. Lysates were probed with the anti-HA or anti-CALM antibody. Shown are dot plots with lines representing the median and interquartile ranges (N = 5 biological replicates per genotype per treatment). A linear model was used to compare mean CALM protein levels among treatment groups within the same genotype and treatment effects between genotypes.