Horseradish peroxidase conjugated anti rabbit igg or anti mouse igg

Whole-cell extracts were made as previously described (Engedal et al, 2002 (link)), resolved by SDS–PAGE and transferred to a PVDF membrane (Bio-Rad). The blotted membrane was blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween (TBS–Tween) for 1 h followed by incubation with primary antibody in TBS–Tween containing 5% bovine serum albumin (BSA) for 14–16 h at 4°C. Antibodies used were against IRE1α (3294S), phospho-PERK (3179S), PERK (3192S) phospho-eIF2α (9721L), eIF2α (9722S), phospho-JNK (9251L), JNK (9252), ATF4 (11815S), cleaved caspase-3 (9661L), PCNA (13110S) (Cell Signaling), XBP-1 (sc-7160), CHOP (sc-7351), PSA (sc-7638), β-actin (sc-58670), GAPDH (sc-47724), β-tubulin (sc-9104) (Santa Cruz), α-tubulin (Sigma-Aldrich), AR (06-680) (Upstate), and phospho-IRE1α (PA1-16927) (Thermo Scientific). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich) secondary antibodies in 5% nonfat dry milk dissolved in TBS–Tween for 1 h at room temperature. ECL Western blotting analysis system was utilized for detection of the immunoreactive bands according to the manufacturer's instructions (Amersham Pharmacia Biotech).

RAW264.7 cells were seeded in 6-well plates (3 × 10⁶ cells/well) and cultured overnight in a CO₂ incubator. Cells were stimulated with 25 ng/ml LPS from K. pneumoniae in the presence of 10 μm SET-M33 in DMEM for 6 h at 37 °C. After incubation, cells were washed twice with PBS and detached with ice-cold PBS. They were then centrifuged, suspended in lysis buffer (20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% cholic acid, and protease inhibitor mixture) and incubated for 30 min on ice. Products of cell lysis were centrifuged at 12,000 rpm for 10 min at 4 °C, and the concentration of proteins in supernatant (cytoplasmic extract) was determined by Bradford assay (Bio-Rad). 20 μg of total proteins was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked in PBS-5% BSA for 1 h at room temperature and then incubated overnight at 4 °C with antibodies specific for NF-κB p65 (Cell Signaling Technology) and for β-actin (Sigma-Aldrich) diluted in PBS-5% BSA-0.1% Tween 20. After washing with PBS-0.05% Tween 20, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich). Signals were detected using Image LAS4010 (GE Healthcare).