Spa 827

The following antibodies were purchased: insulin (L6B10, Cell Signaling Technology, Danvers, MA, USA and sc-9168, Santa Cruz, California CA, USA), GM130 (cis-Golgi network staining, 610822, BD Transduction Laboratories, New York, NY, USA), FLAG (F-1804, Sigma, St Louis, MO, USA), HA (anti-HA high affinity 3F10, Roche, Basel, Switzerland), KDEL (ER staining, SPA-827, Stressgen, Victoria, BC, Canada), and Alexa Fluor-conjugated secondary antibodies (Thermo Fisher, Rockford, IL, USA). Mouse pancreas-derived NIT-1 cells (purchased from ATCC^® CRL-2055^TM, Manassas, VA, USA) were maintained as described previously^{18 (link)}. Briefly, the cells were plated at a density of 1.5–3.0 × 10⁶ cells/60-mm dish, and the medium (F-12K, Kaighn’s Modification of Ham’s F-12 Medium, 2 mmol/l L-arginine and 7 mmol/l glucose) after 48 h of culture medium was exchanged to fresh medium after 48 h of culture.

Western blots were performed with cell lysates, as well as renal tissue lysates, all in triplicate. 4X SDS lysis buffer with protease and phosphatase inhibitors was used for cell or tissue lysis. A protein assay was performed prior to Western blotting (DC Protein Assay; BioRad). A 15% SDS-PAGE gel was used to probe for acetyl H3, and 10% SDS-PAGE gels were used for all other proteins. Primary antibodies were detected using a horseradish peroxidase-bound secondary antibody and enhanced chemiluminescence. Acetyl H3 (#06–599; Millipore) was used at a dilution of 1:1000, CHOP (sc-7351; Santa Cruz) was used at a dilution of 1:200, β-actin (#66009–1; ProteinTech) was used at a dilution of 1:5000, and GAPDH (#2118; Cell Signaling) was used at a dilution of 1:1000. GRP78 was measured using a KDEL antibody (SPA-827; Stressgen), which binds to the KDEL amino acid sequence in GRP78. It was used at a dilution of 1:1000. Anti-mouse and anti-rabbit secondary antibodies were used at 1:5000 (BioRad). Results were densitometrically quantified using ImageLab software, and each protein was expressed as a ratio of β-actin or GAPDH loading control.