Enhanced chemiluminescence ecl kit

Food-grade Dictyophora was provided by Zhijin Sifang Hongye (Zhijin City, Guizhou Province, China). Sodium arsenite (NaAsO₂) was obtained from Sigma Chemical Corp (St. Louis, MO, United States). Human normal hepatocytes (L-02 cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 cell culture medium, trypsin, and fetal bovine serum (FBS) were purchased from Gibco Company (California, United States). Phosphate buffer saline (PBS) was purchased from Zhongsha Jinqiao Biotechnology Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) was obtained from Sigma (St. Louis, MO, United States). The BCA protein detection kit, protein sample buffer, and Western blot analysis gel preparation kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Protein molecular weight markers were obtained from Fermentas (Burlington, Canada). Polyvinylidene fluoride (PVDF) film and enhanced chemiluminescence (ECL) kit were purchased from Bio-Rad (California, United States). Cell Counting Kit-8 (CCK-8), RIPA lysis buffer, rabbit anti-human Bcl-2, Bax, β-actin, GAPDH, ribosomal protein S5 (Rps5), and 14-3-3 protein sigma (SFN) antibodies and horseradish peroxidase (HRP) labeled secondary antibodies were purchased from Boster Biological Technology, Ltd. (Boster, Wuhan, China).

Mouse macrophage cell line RAW 264.7 was obtained from West China School of Pharmacy Sichuan University (Chengdu, China). RPMI-1640, phosphate buffered saline (PBS) and penicillin/streptomycin was purchased from Hyclone (USA). Fetal bovine serum (FBS) was purchased from Gibco (USA). Cell counting kit-8 (CCK-8) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Mammalian Cell Lysis Reagent was purchased from Fermentas (USA). β-Actin antibody, P62 antibody, goat-anti-mouse-IgG-HRP and goat-anti-rabbit-IgG-HRP were purchased from Santa Cruz (USA). LC3 antibody was purchased from NOVUS (USA). Enhanced chemiluminescence (ECL) kit and PVDF membrane were purchased from Bio-Rad (USA). LPS (lipopolysaccharide) was purchased from Hycult Biotech (Netherlands).

4-phenylbutyrate (4-PBA) was purchased from Sigma Co. (Sigma-Aldrich, St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM), F12 and Fetal Bovine Serum (FBS) were purchased from Invitrogen (Invitrogen, Carlasbad CA). Primary antibodies against GRP78, CHOP, XBP-1, TNF-α, IL-6, caspase-12, Lamin B, GAPDH, IκB-α, NF-κB p65, ATF-6, p-JNK, JNK and IgG-HRP were purchased from Cell Signaling Technology, Inc. (Shanghai, China). A 3.3-kb cDNA fragment (dominant-negative type) encoding HA-tagged JNKK2 (DM)-JNK1 fusion protein, in which lysine 149 in the ATP domain of the JNKK2 moiety was replaced by methionine, and vector cDNA (control) were used, as previously described. Enhanced chemiluminescence (ECL) Kit was purchased from Bio-Rad (Hercules, CA). STZ, thapsigargin, tunicamycin, JNK inhibitor SP600125, and all of the other reagents were purchased from Sigma Co. and otherwise specified.

DMEM and foetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant human bFGF was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Akt, p-Akt (Ser473), anti-ERK1/2, p-ERK1/2 (Thr202/Tyr204), anti-cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, cleaved-PARP, cytochrome c, anti-CHOP, cleaved-caspase-12, glucose-regulated protein (GRP-78), ATF-6 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat antirabbit and antimouse IgG-HRP were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad (Hercules, CA, USA). TBHP, PI3K/Akt inhibitor LY294002, ERK1/2 inhibitor PD98059 and all other reagents were purchased from Sigma-Aldrich unless otherwise specified.

Tunicamycin (TM) and sodium phenylbutyrate (4-PBA) were purchased from Solarbio (Beijing, China). Antibodies against p-PERK andp-IRE1α were from Affinity Biosciences (Zhenjiang, China); GRP78 and CD31, from Abcam (Cambridge, UK); and CHOP, PCNA, and GAPDH, from Proteintech group (Wuhan, China). The secondary antibodies (goat anti-rabbit and anti-mouse) were purchased from Beyotime (Shanghai, China). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad (Hercules, CA, USA).

The kidney lysates were collected using a buffer containing 25 mM Tris-HCl pH7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS with protease inhibitor (Thermo Fisher Scientific, Waltham, MA). The protein concentration was measured with the Pierce Bicinchoninic acid reagent (Thermo Fisher Scientific, Waltham, MA). An equal amount of protein was loaded for SDS-PAGE and transferred onto PVDF membrane. The membranes were then incubated sequentially with a blocking solution containing 5% nonfat milk, primary antibody incubation and finally horseradish peroxidase-conjugated secondary antibody. The antigens on the blots were revealed using the enhanced chemiluminescence (ECL) kit (Bio-Rad, Hercules, CA) to record signals by ChemiDoc MP imaging system (Bio-Rad, Hercules, CA). The densitometry of the blots was performed with Image Lab Software Ver 6.1.0 (Bio-Rad, Hercules, CA).