Figure 2. ER stress induces Bax and Bak... | Rockefeller University Press

Figure 2.

$ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 µM) or tunicamycin (Tuni; 5 µg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 µg/ml), Thap (2 µM), or Tuni (10 µg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 µg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.$

ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 µM) or tunicamycin (Tuni; 5 µg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 µg/ml), Thap (2 µM), or Tuni (10 µg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 µg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.