Complete mini protease inhibitor cocktail inhibitors

Cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) with cOmplete Mini Protease Inhibitor Cocktail inhibitors (Sigma), and protein levels quantified using Qubit 3.0 Fluorometer (Thermo). Twenty micrograms of protein was denatured and loaded on a 4–20% gradient gel (BioRad), and then transferred to PVDF membranes at 4°C. Membranes were blocked in Casein + 0.1% Tween-20 (Thermo), and incubated overnight at 4°C with the following antibodies with agitation: MYOD monoclonal antibody 5.8A at 1:1000 (Thermo), MYC 71D10 monoclonal antibody at 1:1000 (Cell Signaling), alpha-Tubulin DM1A monoclonal antibody at 1:1000 (Cell Signaling), and MF20 at 1:1000 (Developmental Studies Hybridoma Bank). Goat anti-mouse IgG (H + L)-HRP conjugate and goat anti-rabbit IgG (H + L)-HRP conjugate secondaries were utilized at 1:20,000 (BioRad). Signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Fisher). Membranes were imaged on the BioRad GelDoc XR + and quantification was performed in ImageJ.

Cells or tumor tissues were harvested and lysed in RIPA buffer complemented with cOmplete Mini Protease Inhibitor Cocktail inhibitors and Phosphatase inhibitor cocktails (Sigma). Lysates were denatured by boiling in 1× Laemmli buffer at 95°C for 5 min and loaded on a 4–20% gradient gel (BioRad). PVDF (polyvinylidene difluoride) membranes were used for proteins wet transfer (BioRad). Membranes were blocked in Casein + 0.1% Tween-20 (Thermo), and incubated overnight at 4°C with the primary antibodies Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370 S, Cell Signaling) at 1:2000, p44/42 MAPK (Erk1/2) (137F5) (4695 S, Cell signaling) at 1:2000, β-Actin Antibody (4967 S, Cell Signaling) at 1:2000, and α-Tubulin Antibody (2144 S, Cell Signaling) at 1:2000. Secondary antibody used were Anti-rabbit IgG, HRP-linked Antibody (7074 S, Cell Signaling) at 1:5000, and Anti-mouse IgG, HRP-linked Antibody (7076 S, Cell Signaling) at 1:5,000. Signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Fisher). BioRad GelDoc XR was used for membranes imaging. The western blot analysis was performed at least three times for each experiment in three technical replicates.