Anti grp78

Tensile tissue or treated fibroblasts were lysed in radioimmunoprecipitation buffer (RIPA) containing protease and phosphatase inhibitors as previously described (Jiang et al., 2014 (link)). A BCA Protein Assay Kit (Thermo Fisher Scientific, IL, USA) was employed to determine protein concentration. Twenty micrograms of lysate were loaded onto a 10 to 15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) for separation and then electrotransferred to a polyvinylidene fluoride membrane (PVDF; Millipore, Bedford, Massachusetts, USA). Membranes were incubated with primary including anti-COL3A1, anti-α-SMA, anti-ATF-6, anti-phospho-IRE1 (anti-p-IRE1), anti-CHOP, anti-Bcl-2 (Abcam, Cambridge, MA, USA), anti-GRP78 (glucose regulated protein 78), anti-Bax, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) and secondary antibody (Abcam, Cambridge, MA, USA). β-Actin was used for standardization. A semi-quantitative analysis was performed using ImageJ software.

Immunoblotting was carried out as previously described [55 ]. The following antibodies were used in this study: anti-PARP-1, anti-cleaved caspase-3, anti-cleaved caspase-8, anti-caspase-9, anti-cleaved caspase-9, anti-HO-1, anti-NOXA, anti-CHOP, anti-GRP78, anti-PUMA (Cell signaling Technology, Beverly, MA), anti-actin (MP Biomedicals, Solon, OH), goat anti-rabbit IgG-horseradish peroxidase (HRP), and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

The plumbous acetate [Pb(Ac)₂] for Pb²⁺ supply was purchased from Sigma–Aldrich. Dulbecco's Modified Essential Medium (DMEM) and FBS were purchased from GIBCO Invitrogen. The Lyso-Tracker Red probes for acidic lysosome staining were from Beyotime. Anti-GRP78 (glucose-regulated protein 78), anti-GRP94, phosphorylates eukaryotic initiation factor 2 (anti-peIF2a), anti-cleaved caspase-3, anti-light chain 3 (anti-LC3) and anti-pS6 were purchased from Cell Signaling Technology. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase), BCL2-associated X protein (anti-Bax), B-cell lymphoma 2 (anti-BcL2) and anti-p70S6K (S6 kinase 1) antibodies were from Millipore. The p62 antibody was from Santa Cruz. Other reagents were of the highest purity available.

Protein extracts were obtained from kidneys using T-PER Mammalian Protein Extraction Reagent (Pierce, Thermo Scientific, Rockford, USA), as indicated by the manufacturer, in the presence of a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, Milan, Italy). Protein content was determined with bicinchoninic acid protein assays (BCA, Pierce, Euroclone, Milan, Italy). An appropriate amount of protein was run on SDS-PAGE under reducing conditions for immunoblotting. The separated proteins were then semi-dry transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and proteins of interest were revealed with specific antibodies: anti-p-S6 (Ser235/236), anti-S6, anti-p-AKT (Ser473), anti-AKT, anti-p-eNOS (Ser1177), anti-eNOS, anti-COX IV, anti-Cyt c, and anti-Grp78 (all from Cell Signaling, Euroclone, Milan, Italy); anti-SIRT1, anti-Grp75, and anti-Bcl-2 (all from Santa Cruz); and anti-PGC-1α (Abcam, Cambridge, UK) each at 1:1,000 dilution. Anti-β-Actin (1:10,000; Cell Signaling) and anti-Vinculin (1:10,000; Sigma-Aldrich) were used as loading controls. Immunostaining was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse immunoglobulin for 1 h at room temperature (Tedesco et al. 2010 (link)). Amounts of each protein were measured using SuperSignal Substrate (Pierce) and densitometrically quantified with an IMAGEJ software image analyzer.

Primary rabbit polyclonal anti-REEP5 antibody (IB: 1:1000 dilution, IF: 1:800 dilution; 14643-1-AP; Proteintech), polyclonal anti-Nogo-A/B (IB: 1:1000 dilution, IF: 1:1000 dilution; PA1-41220; ThermoFisher), polyclonal anti-ATL3 antibody (IB: 1:1000 dilution, IF: 1:800 dilution; PA5-24652; ThermoFisher), polyclonal anti-CKAP4 antibody (IB: 1:1000 dilution, IF: 1:800 dilution; PA5-42926; ThermoFisher) and mouse monoclonal anti-Alpha sarcomeric actinin antibody (IF: 1:500 dilution; MA1-22863; ThermoFisher), monoclonal anti-Triadin antibody (IF: 1:500 dilution; MA3-927; ThermoFisher), monoclonal anti-RyR2 antibody (IF: 1:500 dilution; ab2827; Abcam) were used for immunocytochemistry and immunoblot studies. Anti-alpha tubulin (#2144), anti-GRp78 (#3177), anti-GRp94 (#2104), anti-GAPDH (#2118) antibodies from Cell Signaling; anti-ATF4 (ab216839), anti-Caspase12 (ab62484) antibodies from Abcam were used for immunoblot studies at 1:1000 dilution. Anti-GFP (sc390394) antibody from Santa Cruz Biotechnology was used for immunoblot studies at 1:1000 dilution.

Anti-Grp78, anti-NF-κB, anti-cleaved caspase 3 (Asp175), anti-phospho-JNK (Thr183/Tyr185) and anti-phospho STAT 3 (Tyr705) antibodies were purchased from Cell Signaling (MA,USA); anti-actin and anti-Brn-3a from Chemicon (MA, USA); anti-CHOP from Santa Cruz Biotechnology (CA, USA); anti-ssDNA from Immuno-Biological Laboratories (Fujioka, JAPAN).