Anti p perk thr981

Cells were washed twice with precooled PBS, and then the total cellular protein was extracted by adding RIPA lysate containing a protease inhibitor, and protein concentration was determined by the BCA method. A volume of 10 ul (4 ug/ul) protein was taken for gel electrophoresis, and the protein was transferred to the PVDF membrane. After being closed with 5% skimmed milk for 1 hour at room temperature, the primary antibody was incubated overnight, including anti-PNPLA3 (ab81874; Abcam), anticleaved caspase3 (#9664, Cell Signaling Technology), anti-BIP (#3183, Cell Signaling Technology), anti-PERK (#5683, Cell Signaling Technology), anti-p-PERK (Thr981) (sc-32577, Santa Cruz), anti-eIF-2a (#9722, Cell Signaling Technology), anti-p-eIF-2a (Ser51) (#9721, Cell Signaling Technology), anti-CHOP (#2895, Cell Signaling Technology), anti-PUMA (#4976, Cell Signaling Technology), anti-BAX (#5023, Cell Signaling Technology), and anti-β-actin (#4970, Cell Signaling Technology). The membrane was then washed with TBST and incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature. Protein bands were visualized with an infrared fluorescence scanning imaging system (Odyssey ® DLx Imaging System, LI-COR Biosciences). Each experiment was repeated a minimum of three times.

The following antibodies were used in the study: anti-GRP78 (sc-1050, Santa Cruz, CA, USA), anti-p-PERK Thr981 (sc-32577, Santa Cruz), anti-eIf2α (sc-133132, Santa Cruz), anti-CRT (sc-166837, Santa Cruz), anti-ERp57 (sc-28823, Santa Cruz), anti-Ub (sc-8017, Santa Cruz), anti-p-Akt1/2/3 (sc-7985, Santa Cruz), anti-GAPDH (sc-32233, Santa Cruz), anti-GFP (sc-8334, Santa Cruz), anti-Lamin B1 (sc-374015, Santa Cruz), anti-CaM (sc-137079, Santa Cruz), anti-CaMKIIγ (sc-1541, Santa Cruz), CREB-1 (sc-186, Santa Cruz), anti-p-CaMKII (ab182647, abcam, Waltham, MA, USA), anti-Bcl-2 (610539, BD Biosciences, Franklin Lakes, NJ, USA), anti-cleaved-caspase 3 (9664, Cell Signaling Technology, Danvers, MA, USA), anti-p-CREB Ser133 (9198, Cell Signaling Technology), anti-p-eIF2α Ser51 (9721, Cell Signaling Technology), and anti-PARP (9542, Cell Signaling Technology). All secondary antibodies (HRP-conjugated anti-rabbit, anti-mouse, and anti-goat) were purchased from Sigma (Sigma-Aldrich Inc., St. Louis, MO, USA). All the drug formulations were purchased from Sigma (Sigma-Aldrich Inc.).

The following antibodies were used in this study: anti-PERK (sc-377400, Santa Cruz, CA, USA), anti-p-PERK Thr981 (sc-32577, Santa Cruz, CA, USA), anti-eIf2α (sc-133132, Santa Cruz, CA, USA), anti-PP1 (sc-7482, Santa Cruz, CA, USA), anti-p-Akt1/2/3 (sc-7985, Santa Cruz, CA, USA), anti-GAPDH (sc-32233, Santa Cruz, CA, USA), anti-cleaved-caspase 3 (#9664, Cell Signaling Technology, Danvers, MA, USA), anti-p-eIF2α Ser51 (#9721, Cell Signaling Technology), and anti-PARP (#9542, Cell Signaling Technology). All the secondary antibodies (HRP-conjugated anti-rabbit, anti-mouse, and anti-goat) and all the drug formulations were purchased from Sigma-Aldrich Inc.