Pvdf transfer packs

The interaction between TasA and oleosin was examined by far-western blotting^{49 (link)}. Purified proteins were separated by SDS–PAGE through a 15% acrylamide gel and electrophoretically transferred onto PVDF membranes using a Trans-Blot Turbo transfer system (Bio-Rad) and PVDF transfer packs (Bio-Rad). The proteins were renatured by incubation of the membrane in denaturing and renaturing buffer (100 mM NaCl, 20 mM Tris, pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween 20, 2% skim milk powder and 1 mM DTT) containing decreasing concentrations of guanidine HCl (6, 3, 1, 0.1 and 0 M) and the membrane was blocked with blocking buffer (TBS containing 0.1% Tween 20 and 5% skim milk). Then, the membrane was incubated in protein-binding buffer (100 mM NaCl, 20 mM Tris, pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween 20, 3% skim milk powder and 1 mM DTT) containing 10 μg of purified TasA at 4 °C overnight. Excess protein was removed, and bound TasA was subjected to immunodetection. The membrane was probed with an anti-TasA antibody (rabbit) at a 1:20,000 dilution in blocking buffer. A secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (Bio-Rad) was used at a 1:3,000 dilution in the same buffer. The membranes were developed using Pierce ECL western blotting substrate (Thermo Fisher).

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.

SDS-PAGE gels were routinely used to analyze protein samples. Precipitated proteins were resuspended in 1× Laemmli sample buffer (Bio-Rad) and heated at 100°C for 10 min. Proteins were separated via SDS-PAGE in 15% acrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo transfer system (Bio-Rad) and PVDF transfer packs (Bio-Rad). For immunodetection of recombinant His-tagged Tse1, the membranes were probed with anti-His antibodies (rabbit; Sigma no. SAB1306085) used at a 1:1,000 dilution in Pierce protein-free (Tris-buffered saline [TBS]) blocking buffer. A secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (Bio-Rad no. 1706515) was used at a 1:3,000 dilution in the same buffer. The membranes were developed using the Pierce enhanced chemiluminescence (ECL) Western blotting substrate (Thermo Fisher). For immunodetection of YFP fused to RsiW, anti-YFP antibodies were used at a 1:1,000 dilution in blocking buffer, and a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase was used at a 1:3,000 dilution in the same buffer.