Trans blot transfer pack nitrocellulose

After 24 h of treatment, both floating and attached cells were recovered. They were then centrifuged for 5′ at 350 g at 4°C and washed twice with 10 ml cold PBS and re-suspended in 1 ml cold PBS. Cells were then pelleted at 500 g and lysed in RIPA buffer with Halt™ Protease and Phosphatase Inhibitor Cocktail (Pierce, ThermoFischer Scientific). The lysates were incubated 30′ on ice and then centrifuged for 15′ at 16,000 g at 4°C. Supernatants were recovered, and protein concentrations were quantified by the Bradford method (Pierce, ThermoFischer Scientific, BCA Protein Assay Kit).
Lysates were prepared at 1-4 μg/μl in sample buffer (5x; 1.47 M sucrose, 10% SDS, 5 mM EDTA, 300 mM Tris pH 8.8, 0.25% Bromophenol blue, and 130 mM dithiothreitol) and the proteins were run on TGX precast 4-20% gels (BioRad, Switzerland), transferred onto Trans-Blot transfer pack nitrocellulose (BioRad, Switzerland), and analyzed by western blotting.

Protein extracts were either loaded separately (for quantifications) or pooled (for figure pictures) for each treatment condition. Proteins were run on TGX precast 4-20% gels (BioRad, Switzerland), transferred onto Trans-Blot transfer pack nitrocellulose (BioRad, Switzerland). Western blots were probed with the following antibodies and concentrations: Primary antibodies (dilution and catalogue number) from Cell Signaling (Purchased from Bioconcept AG, Switzerland): ERK1/2 (1:5000 9107); P-ERK1/2 (1:2000 4370); pan-AKT (1:2000 4691); pan-Akt (1:2000 6040); P-AKT-Ser473 (1:2000 4060) and b-actin (1:5000 or 1:10’000 A2066). Secondary antibodies, Li-Cor Biosciences (Bad Homburg Germany): IRDye 680RD Goat anti-Mouse IgG (H + L) (1:10’000, 926-68071), IRDye 800CW Goat anti-Rabbit IgG (H + L) (1:10’000, 926-32210). Blots were scanned using a Li-Cor ODYSSEY Sa fluorescent western blot scanner and quantified with the ODYSSEY Image Studio software.

Phosphate Buffered Saline (PBS) without Ca²⁺ and Mg²⁺ was purchased from Biochrom (Berlin, Germany). Gels (4–20%, Mini-PROTEAN), molecular weight markers (Precision plus Western C protein standard), and reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), membranes (Trans-Blot transfer pack, nitrocellulose), as well as reagents for Western Blotting and the DC Protein Assay kit were obtained from Bio-Rad (Hercules, CA, USA). Annexin V binding buffer was from Becton Dickinson (Franklin Lakes, NJ).