TMX1 thioredoxin and palmitoylation motifs regulate interaction with SERCA2b. (A) TMX1-SERCA2b coimmunoprecipitation. A375P cells were transfected with mycSERCA2b. DSP cross-linked lysates (5% inputs, left) and myc immunoprecipitates (IP) were analyzed for calnexin (CNX) and coimmunoprecipitating endogenous TMX1. The signal of TMX1 immunoprecipitated together with mycSERCA2b was expressed relative to the background. n = 3; ***, P = 0.001 (standard error expressed vs. Mock). (B) TMX1 does not disrupt calnexin–SERCA2b interaction. Control and TMX1-transfected HeLa cells were transfected with mycSERCA2b. DSP-cross-linked lysates (5% inputs, left) and myc immunoprecipitates were analyzed for calnexin and coimmunoprecipitating endogenous TMX1. (C) TMX1-SERCA2b interaction depends on calnexin. Calnexin WT and KO MEFs were transfected with mycSERCA2b and FLAG-tagged TMX1 as indicated. DSP cross-linked lysates (5% inputs, left) and myc immunoprecipitates were analyzed for coimmunoprecipitating FLAG-tagged TMX1. n = 4; **, P = 0.0114 for immunoprecipitation (standard error expressed vs. wild type). (D) Analysis of TMX1 heterozygous KO and shTMX1-transfected HeLa and A375P lysates. TMX1 heterozygous KO HeLa cells were generated with the CRISPR/Cas9 method. Their lysates and control lysates were probed with our anti-TMX1 antibody. For KD, HeLa and A375P cells were stably transfected with shTMX1 and probed with an anti-TMX1 antibody. (E) Calnexin-SERCA2b interaction depends on TMX1. TMX1 WT and KO HeLa cells were transfected with mycSERCA2b. DSP cross-linked lysates (5% inputs, left) and myc immunoprecipitates were analyzed for coimmunoprecipitating calnexin. n = 3; **, P = 0.005 (standard error expressed vs. HeLa). (F) Measurement of global ER Ca²⁺ uptake. HeLa heterozygous KO cells were cotransfected with a plasmid encoding ER-targeted aequorin. Luminescence was plotted from three independent experiments. *, P = 0.02 (standard error expressed vs. HeLa). (G) ER Ca²⁺ uptake in intact cells. HeLa heterozygous KO cells were cotransfected with a plasmid encoding LAR-ER-GECO. Ca²⁺ was depleted with reversible tert-BuBHQ and Ca²⁺ was added back at 1 mM in tert-BuBHQ–free medium. Relative fluorescence increase was plotted and quantified from four independent experiments (P = 0.06; standard error expressed vs. HeLa). (H) ER Ca²⁺ leak in intact cells. HeLa heterozygous KO cells were cotransfected with a plasmid encoding LAR-ER-GECO. Ca²⁺ was depleted with reversible tert-BuBHQ. Relative fluorescence decrease was plotted and quantified from seven independent experiments (*, P = 0.03; standard error expressed vs. HeLa).