Immun blott pvdf membrane

Tissue lysate containing 30 μg protein was separated by SDS–PAGE and transferred to Immun-Blott PVDF membrane (GE Healthcare). The membrane was blocked in 5% milk for 1 h and then incubated with primary antibody (1:1000 dilution) of p53, phospho p53, Bax, Bcl-2, caspase 3, cleaved caspase 3, PARP, p44/p42 MAPK, phospho p44/p42 MAPK, c-Myc, phospho (serine 62) c-Myc, p21 ^waf1/cip1, cyclin D1, phospho Rb (807/811) (Cell Signalling Technologies), anti-GTPase Hras (Abcam), respectively, as needed. GAPDH (Biobharati, India) was used as a loading control. After washing three times with TBS-T (20 mM Tris, 500 mM NaCl, 0.1% Tween-20, pH 7.5), the membrane was incubated with HRP-conjugated secondary antibody (1:3000 dilution) for 1 h at room temperature. The membrane was further washed with TBS-T and developed with Lumiglo reagent (Cell Signaling Technologies).

Renal pelvis tissue lysate (30 μg protein) was separated by SDS–PAGE and transferred to Immun-Blott PVDF membrane (GE Healthcare). The membrane was blocked in 5% skim milkin TBS for 1 h and then incubated with primary antibody (1:1000) dilutions of phospho p53(Cell Signalling Technologies; CST#2527 overnight at 4 °C), Bax(CST#2774 overnight at 4 °C), Bcl-2(CST#2872 overnight at 4 °C), caspase 3(CST#9662 overnight at 4 °C), cleaved caspase 3(CST#9661 overnight at 4 °C), erk1/2(CST#4695 overnight at 4 °C), phospho-erk1/2(CST#4370 overnight at 4 °C), c-Myc (CST#13987 overnight at 4 °C), p21 waf1/cip1(CST#2947 for 5 h at 37 °C), cyclin D1(CST#2987 for 5 h at 37 °C), phospho pRb (807/811)(CST#8516 overnight at 4 °C), anti-GTPase Hras(1:3000 dilution) (Abcam#ab201054 overnight at 4 °C). cyclin E2 (1:2000 dilution) (Abcam #ab32103 overnight at 4 °C). GAPDH (1:3000)(Biobharati, India overnight at 4 °C) was used as a loading control. After washing three times with TBS-T (20 mM Tris, 500 mM NaCl, 0.1% Tween-20, pH 7.5), the membrane was incubated with the goat anti-rabbit/mouse HRP-conjugated secondary antibody(Genei, Bangalore (1:3000 dilution) (for 1 h at room temperature. The membrane was further washed with TBS-T and developed with Lumiglo reagent (CST).