Figure 2: R1346H affects surface expression of hCaV3.3.
From: A rare schizophrenia risk variant of CACNA1I disrupts CaV3.3 channel activity
All membranes were cut in two; upper membranes were probed with anti-FLAG to measure hCaV3.3 levels and lower membranes with control antibodies. (a) Anti-FLAG signals from Flp-In T-REx HEK293 cell lysates after induction for 24, 48, and 72 hrs with (lanes 1–3), and without (lanes 4–6), 1 μg/ml doxycycline. (b) Anti-FLAG signals in whole cell lysates from cells expressing WT-hCaV3.3, untreated (1), with glycosidase (2), and same as (2) but lacking glycosidase (3). (a, b) Compiled figures from 4 digital images of the same gel. Protein ladder images are juxtaposed to the immunoblots; ladder lane is colored in blue. Dotted lines indicate the spliced borders of two immunoblots. (c) Anti-FLAG hCav3.3 levels in whole cell lysate (1–3) and biotin-surface fraction (4–6) from cells expressing WT (1, 4), T797M (T/M) (2, 5), and R1346H (R/H) (3, 6). (d) Anti-FLAG signal in whole cell and biotinylated (surface) preparations from cells expressing T/M and R/H shown relative to WT and normalized to controls (cadherin and β-actin). Mean ± SE values for T/M were 1.12 ± 0.08 (n = 18, whole cell) and 0.87 ± 0.13 (n = 7, surface); for R/H were 0.29 ± 0.03 (n = 18, whole cell) and 0.12 ± 0.03 (n = 7, surface). Coefficient of variation: T/M, 31% and R/H, 40% (1000 samples bootstrapping). (e) Anti-FLAG signals in biotinylated surface protein from cells expressing WT-hCaV3.3, untreated (1), glycosidase exposure (2) and, conditions same as (2) but lacking glycosidase (3). (f) Average fraction of upper MW band relative to total hCaV3.3 for WT: 0.80 ± 0.02 (n = 8); T/M: 0.80 ± 0.02 (n = 8); and R/H: 0.62 ± 0.05 (n = 8) from data in (c,d). (g) RT-qPCR analysis of hCaV3.3 mRNA 72 hr after doxycycline induction expressed as fold-change relative to non-induced. Each individual point is a separate qPCR measure for 3 biological replicates and 3–4 technical replicates. Lines connect the relative levels of mRNA for three genotypes within each biological experiment. Data do not violate D’Agostino-Pearson test for normality.
