Phospho ire1α pa1 16927

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).

Each of the primary antibodies were used according to the manufacturer’s protocol: Phospho-JNK #4671, JNK #9252, IRE1α #3294, PERK #5683, phospho-p38 MAPK #9215, p38 MAPK #9212, phospho-p44/42 MAPK #9106, p44/42 MAPK #4695, (Cell Signaling, Danvers, MA). phospho- IRE1α #PA1-16927, phospho-PERK #MA5-15033, and HCV core (Thermo Scientific, Rockford, IL). ATF6 #ab11909 (Abcam, Cambridge, MA).