Anti phospho perk

Proteins were isolated from atrial myocardium samples of patients and mice by M‐PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc) that contained protease inhibitors and phosphatase inhibitors. Primary anti‐GRP78 (sc‐376768, Santa Cruz), anti‐CHOP (#2895, Cell Signaling Technology), anti‐GAPDH (Bioworld, AP0063), anti‐phospho‐PERK (sc‐32577, Santa Cruz), anti‐PERK (sc‐13073, Santa Cruz), anti‐TGF‐β(Abcam, ab92486), anti‐α‐SMA(Abcam, ab32575), anti‐Collagen I (Abcam, ab34710) and anti‐Collagen III(Abcam, ab7778) were used for Western blotting experiments. Gel Imaging System (Tanon) and Image J software were used to image and analyse Western blotting bands.

Whole cell lysates were prepared and analyzed by Western blotting as described previously [32 (link)]. Proteins in cell lysates were separated by precast 8-20% SDS-polyacrylamide gel electrophoresis, and then electrophoretically transferred from the gel onto polyvinylidene difluoride membranes. After blocking, blots were incubated with anti-GRP78, anti-GRP94, anti-phospho-PERK, anti-caspase-12, anti-caspase-7, anti-CHOP, anti-PARP, and anti-β-actin antibodies (Santa Cruz Biotechnology) and anti-eIF2α (Cell Signaling Technology, Danvers, MA, USA) in PBS within 0.1% Tween 20 for 1 h followed by two 15 min washes in PBS with 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min. Detection was performed with Western blotting reagent ECL (Amersham-GE Healthcare Life Sciences, Pittsburgh, PA, USA), and chemiluminescence was exposed by the Kodak X-Omat films.

Anti-HDAC6 (sc11420, Santa Cruz), anti- acetyl-histone H3 (9649, Cell Signaling Technology), anti-BAX (ab32503, Abcam), anti-Bcl-2 (ab3214, Abcam), anti-cleaved caspase-3 (9664, Cell Signaling Technology), anti-phospho-JNK (4668, Cell Signaling Technology), anti-JNK (ab208035, Abcam), anti-phospho-eIF2α (3398, Cell Signaling Technology), anti-eIF2α (5324, Cell Signaling Technology),anti-phospho-PERK (sc32577, Santa Cruz) anti- GRP78 (ab108613, Abcam), anti- IRE1α (ab48187, Abcam), anti-CHOP (2895, Cell Signaling Technology), anti-ATF4 (11815, Cell Signaling Technology), anti- XBP1 (ab37152, Abcam), anti-caspase-12 (ab62484, Abcam).

HaCaT cell lysates were prepared, and total protein was extracted and separated by SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane and the membrane was incubated with the following primary antibodies: Anti-CHOP, anti-IRE1, anti-caspase-12, anti-poly (ADP-ribose) polymerase (PARP), anti-Mcl-1, anti-caspase-3, anti-caspase-9 (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-IRE1 (Abcam, Cambridge, MA, USA), anti-phospho-PERK, anti-PERK, anti-phospho-eIF2, anti-glucose-regulated protein, 78 kDa (GRP78), anti-XBP-1, anti-ATF6, anti-Bcl-2, anti-Bax (Santa Cruz Biotechnology), and anti-actin (Sigma Aldrich). Subsequently, goat anti-rabbit or mouse IgG secondary antibodies conjugated with horseradish peroxidase (Invitrogen) were added and incubated. Protein expression was detected using Amersham enhanced chemiluminescence western blotting detection reagent (GE Healthcare, Buckinghamshire, UK).

Antibodies to detect human pancreatic ER kinase (PERK), eukaryotic translation initiation factor 2a (eIF2a), activating transcription factor (ATF)-3, ATF-4, C/EBP homologous protein (CHOP), cation transport regulator homolog 1 (CHAC1), and caspase 12 were purchased from Cusabio (Hubei, China). Anti-phospho-PERK, p-eIF2a, glutathione peroxidase 8 (GPX8), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). MS-275, suberanilohydroxamic acid (SAHA), tertiary-butyl-hydroperoxide (tBHP), tunicamycin, and thapsigargin were purchased from Tocris Bioscience (Bristol, UK). Cell-Counting Kit 8 (CCK8) was purchased from Dojindo Molecular Technologies (Rockvill, MD, USA). The apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). GPX8 rescue PLAK1 was a gift from Prof. Yoav Shaul (Addgene plasmid #161517).

Immunoblot analysis was performed according to a previously described method48 (link). The following primary antibodies were used: anti-phospho-IRE, anti-IRE1 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-PERK, anti-PERK (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GRP 78 antibody (1:200; Abcam, Cambridge, MA, USA), anti-CHOP antibody (1:1000; Abcam, Cambridge, MA, USA), anti-cleaved caspase-12 (1:200; Abcam, Cambridge, MA, USA), anti-AdipoR1 antibody (1:1000; BD Bioscience, Franklin Lakes, NJ, USA), anti-AdipoR2 antibody (1:1000; BD Bioscience, Franklin Lakes, NJ, USA), anti-β-actin antibody (1:10000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-tubulin antibody (1:10000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then washed and incubated with suitable secondary antibodies for 1 h at room temperature. Antigens were detected using the chemiluminescence technique (Amersham Pharmacia Biotech Piscataway, NJ, USA). Image analysis was completed using the Bio-Rad Laboratories (Hercules) analysis software.