Anti rabbit or anti mouse igg secondary antibodies

Total protein extracted from podocytes was lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), and 0.5% deoxycholic acid sodium salt (DOC)). Equal amounts of protein were subjected to 8–12% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (Amersham Life Science, USA). After blocking with 5% non-fat milk in PBS containing 0.05% Tween-20 (PBST) for 1 h at room temperature, the membranes were incubated overnight at 4°C with one of the following primary antibodies: rabbit anti-nephrin (Sigma, USA), rabbit anti-calcineurin (Abcam, USA), rabbit anti-α-spectrin (Abcam, USA), rabbit anti-calpain 1 (Abcam USA), mouse anti-β-tubulin (Santa Cruz Biotechnology, USA), or mouse anti-β-actin (Santa Cruz Biotechnology, USA). The membranes were then rinsed 3 times for 8 min each in PBST and incubated with anti-rabbit or anti-mouse IgG secondary antibodies (Santa Cruz Biotechnology, USA). After a final washing step, the membranes were developed using an enhanced chemiluminescence reagent (Millipore, USA), and protein bands were scanned. Finally, images were collected and analyzed using ImageJ software (National Institutes of Health, USA).

Protein was extracted from AGS cells after indicated treatments. After calculating protein concentration, equal amounts of the SDS-PAGE-separated lysates were transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently blocked at room temperature with 1 × TBS containing 0.1% Tween 20 and 5% skim milk. The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-survivin, anti-HSP27, anti-HSP70, anti-HSP90, anti-p-ERK (Thr202/Tyr204), anti-ERK, anti-p-p38 (Thr180/Tyr182), anti-p38, anti-p-JNK (Thr183/Tyr185), anti-JNK (Cell Signaling Technology, Danvers, MA, USA), anti-β-actin, anti- Bcl-xL, anti-Bcl-2, anti-cyclin B1, anti-cyclin D1, anti-MMP9, anti-MMP2, anti-VEGF (Santa Cruz Biotechnology, Inc., Dallas, TEX, USA), anti-HSF1, anti-pHSF1 (Abcam, Inc., Waltham, MA, USA), and anti-cleaved caspase (Genetex, Irvine, CA, USA). The membranes were washed three times before exposure to diluted anti-rabbit or anti-mouse IgG secondary antibodies (Santa Cruz Biotechnology, Inc.) for an hour at room temperature. The blots were washed thrice with 1 × TBS-T buffer for 10 min between each stage. The membranes were identified using enhanced chemiluminescence (Millipore, Billerica, MA, USA).