Enhanced chemiluminescence assay

Cells were lysed using preheated 2% SDS by vortexing vigorously for 2–3 s at maximum speed, followed by boiling for 30 min. The same amount of proteins were subjected to SDS-PAGE, transferred to PVDF membranes (Immobilon-P; EMD Millipore), and blocked for 1 h at RT with 3% milk in 1× Tris-buffered saline Tween-20 (TBST; 25 mM Tris, 150 mM NaCl, and 2 mM KCl, pH 7.4, supplemented with 0.2% Tween-20). Blotting was performed with primary antibodies at 4°C overnight. After washing the membranes with TBST three times for a total of 30 min, HRP-conjugated secondary antibodies were incubated at RT for 1 h. The membranes were washed for 30 min with TBST three times, and proteins were visualized with an enhanced chemiluminescence assay (Thermo Fisher Scientific) or Femto chemiluminescence assay (Thermo Fisher Scientific).

Cells were grown at a density of 7.5×10⁵/ml for 24 h. Cells were collected, washed with cold PBS, and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors [26] (link), [28] (link), [29] (link). Protein estimation was performed using Bradford reagent. Equal amounts of protein were electrophoresed in a 12% reducing SDS-PAGE gel. Proteins were transferred to PVDF membranes; non-specific binding was blocked with 5% non-fat milk in TBST buffer (4 mM Tris base, 10 mM NaCl, pH 7.5, 0.1% Tween-20), and incubated with the indicated primary antibody at 4 °C overnight. Antibodies against CuZnSOD (gift from Dr. Oberley, University of Iowa, IA), poly ADP-ribose polymerase (PARP, full-length, Cell Signaling Technology, Danvers, MA), and actin (Cell Signaling Technology) were used. Blots were then incubated for 1 h at RT with HRP-tagged secondary antibody and developed using an enhanced chemiluminescence assay (Thermo Scientific, Waltham, MA). Bands were visualized by autoradiography and protein expression was quantified using ImageJ 1.38× software (http://rsbweb.nih.gov/ij/index.html).

Immunoblotting was performed as previous described [21] (link). Briefly, frozen liver tissues were homogenized for dissecting in RIPA lysis buffer added with protease inhibitors (Sigma). After centrifuging, the supernatant was collected and incubated in 100 ℃ with the addition of loading buffer for 10 min. When firstly separated by SDS-PAGE, proteins in the gel were transferred to polyvinylidene difluoride (PVDF) membrane. Subsequently, the membranes were incubated in 10% fat-free milk for 1 h. For primary antibodies incubation, membranes were respectively subjected to an overnight incubation with the antibodies against p-IRE1α (1:500, Novus Biologicals), IRE1α (1:2000, Cell Signaling Technology), p-eIF2α (1:1000, Cell Signaling Technology), eIF2α (1:2000, Cell Signaling Technology), p-ATK (1:1000, Cell Signaling Technology), AKT (1:2000, Cell Signaling Technology) or GAPDH (1:5000, Abcam) at 4 ℃, following with incubation in horseradish peroxidase-conjugated secondary antibodies. Proteins were detected by enhanced chemiluminescence assay (Thermo Fisher Scientific).