Figure 3: Glycosylated and non-glycosylated hCaV3.3 signals have different decay time courses after inhibition of protein translation.
From: A rare schizophrenia risk variant of CACNA1I disrupts CaV3.3 channel activity
(a) Anti-FLAG signals from total cell lysates at different time points following exposure to 0.8 μg/mL puromycin from cells expressing WT, T797M (T/M) and R1346H (R/H). All membranes were cut in two; upper membranes were probed with anti-FLAG to measure hCaV3.3 levels and lower membranes with anti-GAPDH for normalization. (b) Time course of anti-FLAG hCaV3.3 > 250kDa (closed symbols) and ~ 250kDa (open symbols) signals normalized to GAPDH and represented relative to pre-puromycin levels for WT (black), T/M (red) and R/H (blue). The >250 kDa glycosylated signals were similar among WT, T/M and R/H except at the 2 hr time point. Mean ± SE at 2hr for WT: 1.42 ± 0.10 (n = 8); T/M: 1.32 ± 0.11 (n = 5); and R/H: 0.85 ± 0.17 (n = 5), at 48 hr for WT: 0.36 ± 0.08 (n = 8); T/M: 0.27 ± 0.06 (n = 5); and R/H: 0.50 ± 0.12 (n = 5). Mean ± SE are shown for each time point. For all analysis, results shown represent at least three experimental replicates and at least two technical replicates. Data do not violate D’Agostino-Pearson test for normality, and comparisons were analyzed by one-way ANOVA with Dunnett’s post hoc test. (c) GAPDH levels during puromycin treatment. The level of GAPDH at each time point is normalized to GAPDH level at time 0 for each condition. Each data point represents mean ± SE for three separate cultures.
