Fig. 2

SEC63 is phosphorylated at Thr537 after ER stress. A HepG2 cells were treated with TM (5 μg/mL) or TG (1 μM) for 8 h. Immunofluorescence was performed with anti-SEC63 antibody. n = 100. B HepG2 cells were treated with TM (left) or TG (right). Cell fraction was performed and SEC63 was evaluated by western blot. C Huh7 cells were treated with TM (5 μg/mL) or TG (1 μM) for 8 h. Immunofluorescence staining was performed. n = 100. D Huh7 cells were treated with TM (left) or TG (right). Then, cell fraction was performed. E The nuclear localization signal sequence of SEC63 was analyzed by JPred4. F Huh7 cells were transfected with GFP-SEC63 or GFP-SEC63 delN. Then, cells were treated with TM and immunofluorescence was performed. Scale bar, 10 μm. G Huh7 cells were transfected as indicated and treated with TM. Cell fraction was performed and SEC63 was evaluated by western blot. H Huh7 cells were treated with TM (5 μg/mL) or TG (1 μM) for 8 h. Cell lysates were subjected to immunoprecipitation and western blot using pan-phos-Serine/Threonine antibody to detect phosphorylation of SEC63. I The phosphorylation sites at SEC63 were predicted by PhosphoSitePlus. J Huh7 cells were transfected with Flag-SEC63 or Flag-SEC63 T537A and then treated with TM (5 μg/mL) or TG (1 μM) for 8 h. Immunoprecipitation was performed and followed by western blot to evaluate the level of SEC63 phosphorylation. K Huh7 cells were treated with TM or TG. Cell lysates were subjected to western blot using anti-phos-T537 (T537-p) SEC63 antibody. L Huh7 cells were transfected with Flag, Flag-SEC63 or Flag-SEC63 T537A. Western blot was performed as indicated. M Huh7 cells were pretreated with KIRA6 (10 μM) for 1 h, followed by TM (5 μg/mL) or TG (1 μM) for 8 h. Cell lysates were subjected to western blot. N Huh7 cells were transfected as indicated. Then, co-immunoprecipitation was performed using the indicated antibody