Anti p65 nf κb c 20

The cells were washed twice with ice-cold PBS and scraped into RIPA buffer plus protein inhibitors. The protein concentration was determined as previously reported by Bradford MM et al., 1976 [33 ]. Proteins were separated on 10 or 12% SDS-PAGE and transferred to polyvinylidene difluoride filters. Membranes were blocked and probed with the following antibodies: anti-phospho-AKT (Thr308) (Cell Signaling), anti-total AKT (Santa Cruz Biotechnology, INC), anti-Tubulin (Sigma), anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-total SAPK/JNK (56G8) (Cell Signaling), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10), anti-p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling), anti phospho-Ser307-IRS1, anti-total IRS1 (Millipore), anti-p65-NF-κB (C-20) (Sigma-Aldrich®), anti-Lamin B Receptor (abcam®) and anti-sXBP1 (R&D biosystems).
The protein band density was measured using the ImageJ 1.45 software (National Institutes of Health, Bethesda, MD). The amount of protein under control conditions was assigned a relative value of 100%.

Podocytes on cover slips were fixed with 4% paraformaldehyde. After blocking, cells were incubated with anti-Paxillin (Calbiochem), phalloidin toxin-Rhodamine (Life Technologies™ R415), anti-p65-NF-κB (C-20) (Sigma-Aldrich®), anti-CHOP (Santa Cruz Biotechnology, Inc.) or anti-GLUT4 (Millipore). Secondary antibodies were FITC-conjugated. Some samples were incubated without the primary antibody to serve as negative controls. The nuclei were visualized using DAPI dye. Photographs were taken using an inverted fluorescence microscope (ECLIPSE 90i, Nikon Instruments Europe B.V.) or confocal microscope LSM710 (Zeiss, Germany). GLUT4 images of podocytes were scored by two independent blinded observers who scored at least 100 cells per condition for cytoplasmic or peripheral localization.