Fig 2.

Mutations in SEC61A1 affect protein stability and disrupt ER/cytosol calcium homeostasis. A, Sec61α1 expression in healthy controls (HCs) and the patient (with P' indicating the patient's status as of April 2019 and P'' indicating the patients status as of June 2018). B, Quantification of (A) normalized to the average of the HCs. C,SEC61A1 mRNA expression in PBMCs (n = 3) and primary fibroblasts from 2 different passages (n = 4), normalized to the average of the HCs. Unpaired t test. D, HL-60 cells transduced with SEC61A1–green fluorescent protein (GFP) stained with anti–protein disulphide isomerase (PDI) (left) and anti-GM130 (right) (n = 2). E, Ratio of cells with colocalization of Sec61α1 and GM130 over the total number of cells from (D), normalized to WT. F, Calcium flux measurements in transduced HL-60 cells. Mean of 3 independent experiments with duplicates. G, Amplitude of calcium efflux following thapsigargin treatment. R represents the ratio of the FuraRed fluorescence intensity at 450/50 nm and 670 nm. Paired t test. H, Area under the curve between t = 60 and 660 seconds (duration of treatment). I,SEC61A1 expression in HeLa cells transfected with small interfering RNA and plasmid as indicated (empty vector [EV]). J and K, Phosphorimaging autoradiogram and quantification of Sec61-dependent cotranslational transport efficiency of Quiescin-sulfhydryl oxidase (K) and Sec61-independent posttranslational transport efficiency of Sec61β (J), normalized to the control (Ctrl). Gly, glycosylated. Ordinary 1-way ANOVA with Sidak correction. Data are represented as means ± SEMs. DAPI, 4',6-Diamino-2-phenylindole.