Anti kdel

Triplicates of 1.5 × 10⁵ cells were washed with PBS and harvested in 50 μl of ice-cold electrophoresis sample buffer. Lysates were boiled for 10 min, separated by electrophoresis using Criterion™ TGX™ Precast 12% gels and transferred to Trans-Blot^® TurboTM Midi Nitrocellulose or PVDF Transfer Packs (Bio Rad, Hercules, CA, USA). For immunoblotting, membranes were blocked in 5% skimmed milk, 5% serum in TBST and proteins detected by specific primary antibodies diluted in TBST containing 5% BSA overnight at 4°C: anti-αII Spectrin (1:1000; Santa Cruz Biotechnology); anti-PARP (1:1000, Cell Signaling, Danvers, MA, USA); anti-caspase-3 (Santa Cruz Biotechnology); anti-peIF2 and anti-eIF2 (1:1000; Cell Signaling); anti-KDEL (1:1000; Stressgen Bioreagents); anti-CHOP (1:250; Santa Cruz Biotechnology). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000, Sigma) in 5% skimmed milk, 1% normal serum in TTBS for 2 h RT and developed using enhanced chemiluminiscence according to the manufacturer’s instructions (Super Signal West Dura, Pierce, Rockford, IL, USA) in a C-Digit^® Blot Scanner (Li-Cor, Lincoln, NE, USA). Signals were quantified using Image Studio™ software (Li-Cor) and values were normalized to β-actin signal and provided as the mean ± SEM of at least three independent experiments.

Equal amounts of protein from lysates were resolved by SDS-PAGE. The following primary antibodies were used: anti-GAPDH (1/20,000 ref. ab8245) anti-KRas (1/500 ref.137739); anti-Nox1 (1/1 000 ref. ab121009); anti-NoxA1 (1/1 000 ref. ab68523); anti-NoxO1 (1/1000 ref. ab34761); anti-Rac1 (1/1000 ref. ab33186); anti-RhoGDI (1/2000 ref. ab53850) from Abcam, anti-GSK3 total (1/1000 ref.5676); anti-pGSK3 (1/1000 ref. 9331) from cell signaling, anti-RhoA (1/1000 ref. SC26C4) from Santa Cruz Biotechnology, anti-GAPDH (1/20,000, ref. G8795) from Sigma, anti-KDEL (1/1000, SPA-827) from stressgen, anti-PDIA1 (1/1000, clone RL90 ref. MA3-019) from Thermo. The HRP-coupled secondary antibodies were purchased from Cell Signaling Technology (1/5000). Fluorescent-coupled secondary antibodies were purchased from Odyssey (1/10,000, anti-mouse ref. 926-32212, anti-rabbit 926-32223, anti-goat ref. 926-32224), and fluorescent immunoblotting were scanned with the Odyssey near-infrared imaging system.