Phenylmethene sulfonylfluoride pmsf 10 mm

Briefly, THP-1 cells were harvested, and lysates were prepared using CytoBuster protein extraction reagent (Novagen/EMD, Gibbstown, NJ) supplemented with a complete mini EDTA-free protease inhibitor (Roche, Basel, Switzerland) and phenylmethene-sulfonylfluoride PMSF (10 mM) (Sigma-Aldrich). Cell lysate protein concentrations were determined, and Western blots were performed as previously described (10 (link)) using α-Yap, α-TEAD (1, 3, and 4), α-Fzd5 receptor, α-BCL-xL, α-Bax antibodies (1:200), α-Caspase -3 and -9 antibodies (1:100), and α-GAPDH (1:10,000). Experiments were performed with three biological and technical replicates, and significance was determined by t-test analysis. All blots are presented in their original form but may have a loss of resolution due to image compression from the dock system, Chemidoc-it2 Imager.

For Western blots, THP-1 cells were harvested and washed twice with PBS and lysates were prepared using CytoBuster protein extraction reagent (Novagen/EMD, Gibbstown, NJ) supplemented with complete mini EDTA-free protease inhibitor (Roche, Basel, Switzerland), phenylmethene-sulfonylfluoride PMSF (10 mM) (Sigma-Aldrich). The cell lysates were centrifuged at 15,000g for 10 min at 4°C. The supernatants were collected, and protein concentration was then measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (15–30 μg/well) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane and immunoblotted with primary antibodies. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (H+L) secondary antibodies (Kirkegaard & Perry Laboratories, Gaithersburg, MD) were used and visualized by SuperSignal West Dura chemiluminescent substrate or ECL (Thermo Fisher Scientific). All Western blots were performed with at least three biological and technical replicates and significance determined by t-test analysis.