Anti bip

Proteins from treated and untreated cell lysates were estimated using the BCA protein estimation assay (Thermo Scientific). Blots were probed for: anti-Sp1; anti-BiP; anti-Ire1α; anti-PERK; anti-phospho-PERK (Thr980); anti-eIF2α; anti-phospho-eIF2α; anti-β-actin (all from Cell Signaling).

For western blot analysis, cells were lysed in lysisbuffer (Cell Signaling) containing Protease Inhibitor Cocktail (Roche #13538100), and sonicated. Samples were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels under reducing conditions and transferred to a PVDF membrane. Specific detection was done by incubating the blot overnight in TBS with 0.1% Tween 20 with 1% BSA. Antibodies used for detection were anti-PERK (Cell Signaling #3192, 1:1000), anti-BiP (Cell Signaling #3183, 1:1000), anti-phospho-eIF2α (Cell Signaling #3398, 1:1000), anti-CHOP (Cell Signaling #2895, 1:1000), anti-XBP1 (Santa Cruz 7160, 1:500), Anti-eIF2α (Cell Signaling #2103, 1:1000), anti-c-Myc (Santa Cruz #764, 1:1000), anti-ATF6 (Bioadacemia 73–500, 1:1000), anti-IRE1α (Cell Signaling #3294, 1:1000), and anti-beta actin (Sigma, A1978, 1:100,000). Secondary antibody detection with HRP labeled polyclonal antibodies was performed (Dako, Goat Anti-Rabbit #P0448, Goat Anti-Mouse #P0447, 1:2000), and antibody visualization was with Lumilight Plus (Roche,12015196001).

Cannabidiol and VAS2870 were purchased from Sigma (St. Louis, MO, USA). TRAIL and anti-DR5 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-Bak, anti-Bcl-2, anti-Mcl-1, anti-Bcl-xL, and anti-DR4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-BIM, anti-survivin, anti-Bid, anti-IRE1α, anti-phospho-IRE1α, anti-Bip, anti-GRP94, anti-ATF6, anti-eIF2α, anti-phospho-eIF2α, anti-CHOP, anti-cleaved PARP, anti-caspase-3, and anti-caspase-9 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). For the secondary antibodies, anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG HRP were purchased from Cell Signaling Technology.

Anti-CHOP, anti-caspase-12, anti-caspase-9, anti-caspase-3, anti-caspase-3 cleaved, anti-Bip, anti-p-eIF2α, anti-p-JNK, anti-p-ERK, anti-p-p38, anti-IRE-1α, anti-Cyt C, and anti-COX IV antibodies were purchased from Cell Signaling Technology; anti-α-tubulin antibodies were acquired from Biyuntian (Nanjing, China); anti-Ag85 and anti-GroEL1 were obtained from Abcam (Cambridge, UK); and anti-BAX antibodies were from Santa Cruz Biotechnology. Goat anti-mouse-IgG-HRP (Proteintech, Wuhan, China) and goat anti-rabbit-IgG-HRP (Proteintech) were used as secondary antibodies. Western blots were detected using the Amersham^TM ECL^TM Prime western blotting detection reagent (GE Healthcare, Buckinghamshire, UK) and the ChemiScope 3400 mini imaging system (Clinx Science Instruments, Shanghai, China). The results shown are representative of three independent experiments.

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.

Reagents used were AZC (TCI Europe, Zwijndrecht, Belgium; # A1043 or Acros Organics, Geel, Belgium; # 105142500), bafilomycin A1 (Sanbio, Uden, The Netherlands; # 11038-500), ethylene glycol tetraacetic acid (Acros Organics; # 409910250), BAPTA-AM (Thermo Fisher Scientific, Waltham, MA, USA; # B6769), TG (Alomone labs, Jerusalem, Israel; # T-650), Fura-2 AM (Life Technologies, Carlsbad, CA, USA; # F1221), staurosporine (LC Labs, Woburn, MA, USA; # S9300), and AMG PERK 44 (Tocris, Abingdon, U.K.; # 5517).
Primary antibodies used were anti-ATF6 (Cell Signaling Technology, Leiden, The Netherlands; # 65880), anti-BiP (Cell Signaling Technologies; # 3183), anti-eIF2α (Cell Signaling Technology; # 9722), anti-phospho-eIF2α (Cell Signaling Technology; # 3398), anti-ERp57 (Cell Signaling Technology; # 2881), anti-ERp72 (Cell Signaling Technology; # 2798), anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA; # G8795), anti-IP₃R (Rbt475 [42 (link)] recognizing all IP₃R isoforms), anti-LC3 (Cell Signaling Technology; # 2775), anti-MCU (Sigma-Aldrich; # HPA016480), anti-PARP (Cell Signaling Technology; # 9532), anti-PMCA (Thermo Fisher Scientific; # MA3-914) recognizing all PMCA isoforms, anti-SERCA2B (Cell Signaling Technology; # 4435), and anti-vinculin (Sigma-Aldrich; # V-9131).

HK-2 cells were lysed in ice-cold RIPA lysis and extraction buffer (Thermo Scientific, USA) containing a protease inhibitor cocktail (Promega, USA). Protein samples (30 μg protein equivalent) were electrophoresed using 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen). The membranes were blocked with 5% milk for 1 h on an orbital shaker, and then incubated overnight at 4 °C with primary antibodies. The antibodies used in the study were as follows: anti-β-Actin (rabbit, 1:1000; Cell Signaling), anti-WFS1 (rabbit, 1:1000; Cell Signaling), anti-BiP (rabbit, 1:1000; Cell Signaling), anti-ATF6 (rabbit, 1:1000; Cell Signaling), anti-XBP1 (rabbit, 1:1000; Cell Signaling) and anti-CHOP (mouse, 1:1000; Cell Signaling). The membranes were washed for three times, followed by incubation with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:10000, [Abcam] or goat anti-mouse, 1:10000, [Abcam]). Enhanced chemiluminescence reagents (Millipore, USA) were used to detect antigen–antibody complexes and the Bio-Rad electrophoresis image analyzer (Bio-Rad, USA) were used to visualize specific bands.