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Fig. 5 HEK293T cells were transfected with plasmids as indicated for 24 h and treated with DMSO or 200 nM ISRIB for 2 h followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, and Endou-Flag using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane. B. HEK293T cells were transfected with plasmids as indicated for 24 h and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 h followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endouc-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane. C. MEF wild-type (WT) and its eIF2αS51A mutant (S51A) cells were treated with 1 μg of TH for 4 h followed by Western blot analysis of p-eIF2α, total eIF2α, and ENDOU. GAPDH served as an internal control. Protein levels relative to each internal control are presented in each lane. D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 h and treated with either DMSO or TH for 4 h followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, Endouc-Flag and ENDOU-1-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. F. HEK293T cells were transfected with negative control siRNA and ENDOU-siRNA for 48 h. After treatment, protein lysates were prepared and subjected to Western blot analysis for ENDOU-1 and CHOP detection using antibodies. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.