Cleaved atf6

We purchased antibodies against Heat shock protein 27 (Hsp27), nucleophosmin (NPM), ATP synthase, and GAPDH from Epitomics (Burlingame, CA, USA) and antibodies against glucose-related protein 78 (GRP78), calreticulin (CALR), protein disulfide isomerase (PDI), cleaved-ATF6, ATF4, and PRDX1 antibodies were purchased from ProteinTech Group (Chicago, IL, USA). Antibodies against Bcl-xl, Mcl-1, Bcl-2, Bad, p-Bad, AIF, cytochrome C, PERK, p-PERK, eIF2α, p-eIF2α, CHOP, caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were obtained from Cell Signaling Technology (Danvers, MA, USA). The protein collection and separation are explained elsewhere [33 (link)]. The primary antibody with appropriate dilution was incubated with the PVDF membrane at 4 °C for 2 h. The membrane was washed three times with PBST (10 mM NaH₂PO₄, 130 mM NaCl, 0.05% Tween 20), and then incubated with the second antibody (goat anti-rabbit or goat anti-mouse IgG and horseradish peroxidase conjugate, 1:5000 in blocking solution) at 4 °C for 2 h. The membrane was then washed three times with PBST. The western blot results were visualized through chemiluminescence by adding ECL Western Blotting Reagents (Pierce).

Rabbit anti-human ERK, p-ERK, JNK, p-JNK, GRP78, ATF4, and cleaved-ATF6 antibodies were purchased from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against AKT, p-AKT, PI3K, p-PI3K, Mcl-1, Bad, p-Bad, Bcl-xl, Bcl-2, Bax, p38, p-p38, PERK, p-PERK, elF2α, p-elF2α, pro-caspase 3, cleaved caspase 3, pro-caspase 9, cleaved caspase 9, cytochrome C, and CHOP were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Cytosolic cytochrome C were separated using a cytochrome C releasing apoptosis assay kit (Biovision, Milpitas, CA, USA).
The flaccidoxide-13-acetate treated sample and DMSO treated control samples (total protein 25 μg) were separated by 12.5% SDS-PAGE, and the proteins on the gel were transferred to a PVDF membrane. The membrane containing transferred protein was blocked in PBS buffer and incubated with primary antibody at 4 °C overnight, followed by secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk) for 2 h at 4 °C. The signals were detected with an enhanced chemiluminescence detection kit.