Anti chop

The lysates of HK-2 cells were prepared with RIPA lysis buffer containing Protease and Phosphatase Inhibitor Cocktail (Cell Signaling Technology, Boston, USA). Then the concentrations of total protein were measured by BCA method (Thermo Fisher Scientific, USA). 30 μg protein were subjected to SDS-PAGE (10%), and then transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Beford, MA, USA). After blocking with5% bovine serum albumin, the membranes were incubated with appropriate primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000, Beyotime, Shanghai, China) at room temperature for 1 h. Following antibodies were used: anti-PERK (1:1000), anti-p-PERK (1:1000), anti-GPR78 (1:1000), anti-CHOP (1:1000), anti-cleaved Caspase12 (1:1000), anti-cleaved Caspase3 (1:1000) (Cell Signaling Technology, USA), anti-GAPDH (1:3000), anti-p-eIF-2α (1:1000), anti-eIF-2α (1:1000), anti-Bax (1:1000), anti-Bcl-2 (1:1000) (Abcam, Cambridge, MA, USA). GAPDH was selected as the loading control. The blots were detected using the enhanced chemi-luminescence (ECL) detection kit (KeyGen Biotech, Nanjing, China).

After treatments, cells were fixed with acetone/methanol (1:1) for 5 min at −20 °C and blocking in 5% BSA in PBS for 30 min. Fixed cells were incubated overnight at 4°C with primary antibody [anti-SDHA (1:500, mouse, Invitrogen), anti-PDI (1:500, rabbit, Stressgen), anti-ATF4 (1:500, rabbit, Santa Cruz Biotechnologies), anti-CHOP (1:500, rabbit, Cell Signaling), anti-cytochrome c (1:500, mouse, BD Transduction Lab.), or anti-COX IV (1:500, mouse, GeneTex)]diluted in PBS and then washed three times in PBS and incubated for 1 h at room temperature with anti-rabbit or anti-mouse Alexa Fluor 488 or 594 (1:1000, Molecular Probes). Slides were mounted with ProLong Gold antifade mounting reagent (Molecular probes) and cell staining was visualized with a fluorescence microscope using Zeiss filter sets #46 and #64HE.

Protein extraction and western blot analysis were performed as previously described [52 (link)]. Anti-Beclin-1, anti-cleaved caspase 7, anti-CHOP, anti-GRP78, anti-phospho-H2AX, anti-LC3, anti-PARP, and anti-p62 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-GRP94 was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Anti-α-tubulin was obtained from Abcam (Eugene, OR, USA).

Tissue samples were homogenized in standard RIPA buffer with PMSF. Protein concentrations were quantified with the Lowry assay using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Then, 30 μg of total protein was diluted to the same volume 2 × SDS-sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 5% 2-mercaptoethanol, and 0.025% bromophenol blue). Cultured cells were lysed in 2 × SDS-sample buffer. Protein lysates were separated by 6–10% SDS-gel electrophoresis, and transferred onto Immobilon-P membranes (Millipore, Billerica, MA, USA). Membranes were blocked in PBST buffer containing 3% skim milk for 1 h at room temperature, probed with primary antibodies and secondary HRP-conjugated antibodies (GE Healthcare, Little Chalfont, UK), and developed using ECL western blot detection reagent (GE Healthcare). Antibodies used in this study are as follows: anti-GADD34 was from Santa Cruz; anti-β-actin was from Sigma; and anti-p-eIF2α, anti-eIF2α, anti-ATF4, anti-CHOP, anti-p-NF-κB p65, anti-NF-κB p65, anti-p-IκBα, anti-IκBα, anti-p-IKKα/β, anti-IKKβ, anti-p-TAK1, anti-p-IRF3, anti-IRF3, anti-p-IKKɛ, and anti-IKKɛ were from Cell Signaling Technology (Danvers, MA, USA).