Mini protean tgx stain free precast polyacrylamide 4 20 gels

Equal quantities of proteins (10 to 30 µg) were run on Mini-PROTEAN TGX Stain Free precast polyacrylamide 4–20% gels (BIORAD) in Tris glycine-SDS buffer (BIORAD). Proteins were transferred on Immobilon-P PVDF membranes (Millipore) in Tris glycine-Ethanol 20% buffer (BIORAD). Stain Free technology allows visualizing total proteins without use of any dye. The following antibodies were used at dilutions from 1/200 to 1/1000: anti-caspase 9 (32539, Abcam, Cambridge, UK), anti-cleaved caspase 3 (5A1E, Cell Signaling, Danvers, MA, USA), anti-PARP (Cell signaling 92845), anti-FGF1 (R&D AB-32-NA, Minneapolis, MN, USA) and anti-FGF1 (Cat.No.010-24161, WAKO, Osaka, Japan), anti-p53 DO1 (Santa Cruz Sc-126), anti-p53pSer15 (Cell signaling 92845), anti-PUMA N-19 (Santa Cruz Sc-19187), anti-Bax I-19 (Santa Cruz Sc-930), anti-p21 C-19 (Santa Cruz Sc-397-G) and anti-TOM40 H-300 (Santa Cruz Sc-11414). Secondary antibodies were HRP coupled (Jackson Immunoresearch, West Grove, PA, USA) and the revelation was performed using Clarity Western ECL Blotting Substrate (BIORAD). Secondary antibodies were HRP coupled (Jackson Immunoresearch) and the detection was performed using Clarity Western ECL Blotting Substrate (BIORAD). The chemiluminescent signal was captured by Chemidoc (BIORAD) and quantification was performed with the ImageLab software (BIORAD).

Equal quantities of proteins (10 to 30 μg) were loaded on Mini-PROTEAN TGX Stain Free precast polyacrylamide 4–20% gels (BIORAD, Hercules, CA, USA) in 1× Tris Glycine-SDS buffer. Proteins were transferred onto Immobilon-P PVDF membranes (Millipore, Darmstadt, Germany) in 1× Tris Glycine with 20% ethanol. Stain-free technology enables the visualization of total proteins adsorbed onto the membrane without the use of any dye. For immunoblotting, antibodies were diluted in TBS-Tween 0.1%. The primary antibodies used for immunoblotting are listed in Table S54. HRP-coupled secondary antibodies were obtained from Jackson Immunoresearch and Diagomics. Chemiluminescent detection was performed with Clarity Western ECL substrate (BIORAD) and the signal was captured by Chemidoc (BIORAD). Quantification was performed using ImageLab software (BIORAD).