Gradient mini precast tgx gel

The SW48, SW48-CR, HCT15 and SW480 cancer cells were seeded into 100 mm³ petri dishes and treated for 24 h with silybin or regorafenib and their combination, as previously indicated. Cells were lysed with RIPA lysis buffer (Sigma-Aldrich, MO, USA) with protease and phosphatase inhibitors cocktail. Protein extracts were then quantified by using Bradford assay (BioRad, CA, USA), according to the manufacturer's instruction. Equal amounts of total proteins were separated by 4–15% gradient mini precast TGX gel (BioRad) and transferred to nitrocellulose membrane (BioRad). The membrane was blocked with 5% of milk at RT for 1 hours and incubated with following primary polyclonal antibodies Caspase 3 (#9662), PARP (#9542), AKT (#9272), p70-S6K (#9205) and monoclonal antibodies Caspase 9 (#9508), p-AKT (#4060) and p4EB-P1 (#2855) purchased from Cell Signaling (Beverly, MA, USA). Monoclonal anti-α-tubulin antibody was provided by Sigma-Aldrich (St. Louis, MO, USA). After incubation with secondary anti-goat antibody at room temperature for 1 hours, according to the manufacturer's instruction, the membranes were developed using an enhanced chemiluminescence (ECL) detection system (Invitrogen, CA, USA). Each experiment was done in duplicate.

Protein lysates were obtained by homogenization in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) with protease and phosphatase inhibitors cocktail (Hoffmann-La Roche). Protein extracts were quantified by using the Bradford assay (Bio-Rad, Hercules, CA, USA) and equal amounts of total protein (40 μg/lane) were separated by 4–15% gradient mini precast TGX gel (Bio-Rad) before transferring to nitrocellulose by standard western conditions, blocked in BSA solution and primary antibodies (1:1000 in BSA solution; incubated overnight at 4 °C). The secondary antibody (1:3000 in 5% milk/TBS/Tween20 solution) was incubated at RT for 1 h before detection. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford, IL, USA) using the ChemiDoc (Bio-Rad). Values were normalized to α-tubulin. Each experiment was performed in duplicate.

H1299 and H460 cancer cells were seeded into 100 mm³ petri dishes and treated for 24 h with pioglitazone [1 and 10 μM]. Protein lysates were obtained by homogenization in RIPA lysis buffer (Sigma-Aldrich, MO, USA) with protease and phosphatase inhibitors cocktail (Hoffmann-La Roche). Protein extracts were quantified by using Bradford assay (Bio-Rad, CA, USA) and equal amounts of total protein (40 μg/lane) were separate by 4–15% gradient mini precast TGX gel (Bio-Rad) before transferring to nitrocellulose by standard western conditions, blocked in BSA solution and primary antibodies (1:1000 in BSA solution; incubated overnight at 4 °C). The secondary antibody (1:3000 in 5% milk/TBS/Tween20 solution) was incubated at RT for 1 h before detection. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford,IL) using the ChemiDoc (Bio-Rad). Values were normalized to α-tubulin. Each experiment was done in duplicate.