Immuno blot polyvinylidene fluoride pvdf membrane

Western blotting was performed using established methodology [22 (link)]. Briefly, cells were washed with PBS and lysed in RIPA buffer (1 X PBS, pH7.4, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1.0 mM sodium orthovanadate, 10 μl of protease inhibitor cocktail (100 x, Calbiochem) per 1 ml of buffer, and 100 μg/ml PMSF). Proteins (20–30 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred on an Immuno-Blot polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% nonfat milk, the membrane was incubated with primary antibodies overnight at 4^°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, 1:3000) and then developed using Enhanced Chemiluminescent (ECL) solution (Pierce). Primary antibodies used were rabbit anti-nephrin (Abcam, 1:1000), goat anti-nAChR α5, α6, α7, β3 receptors (Santa Cruz, 1:1000), rabbit anti-cleaved caspase-3 (Cell Signaling, 1:1000), rabbit anti-Bax (Santa Cruz, 1:1000), rabbit anti-Bcl-2 (Santa Cruz, 1:1000), and goat anti-actin (Santa Cruz, 1:3000). For protein expression quantification, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image/).

Renal cortices were homogenated with lysis buffer and 1x protease inhibitor cocktail in a tissue homogenator (Roche Diagnostics, Indianapolis, IN, USA) and then centrifuged. The supernatant was extracted to measure the concentration of protein. The protein samples were then added in 5x SDS-PAGE loading buffer and were heated at 100°C for 10 min to be denatured. Proteins (80 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to an immunoblot polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking in PBS/Tween (0.1%) with 5% skim milk for 2 hours, the membrane was incubated with primary antibodies (nephrin antibody 1 : 500, podocin antibody 1 : 2000, and CD2AP antibody 1 : 1000) overnight at 4°C. Target bands were detected using a horseradish peroxidase-conjugated secondary antibody and developed using Pierce enhanced chemiluminescence (ECL) (ThermoScientific, Fisher Scientific, Pittsburgh, PA, USA). Band intensity of nephrin, podocin, CD2AP, and VEGF-A was normalized to that of β-actin and expressed as a relative ratio. Semiquantifications were performed using Image J program (National Institutes of Health, Bethesda, MD, USA).