Nb100 91273

SDS-PAGE and western blot (WB) were performed according to standard protocols. Briefly, after respective transfection and LIPUS treatments, hMCSs cells were lysed in lysis buffer containing 15 mM Tris/HCl pH 7.5, 120 mM NaCl, 25 mM KCl, 1 mM EDTA, 0.5% Triton X100, and a Halt Protease Inhibitor Single-Use cocktail (100×, ThermoFisher Scientific, Rodano, Italy). Whole lysate (15 µg per lane) was separated using 4–12% NovexBis-Tris SDS-acrylamide gels (Invitrogen, Life Technologies), electro-transferred on nitrocellulose membranes (Bio-Rad Laboratories Srl, Segrate, Milan, Italy), and immunoblotted with the appropriate antibodies. Antibodies against the following proteins were used: HIF-1α (anti-rabbit HIF-1α, Merck Millipore SpA, Vimodrone, Milan, Italy), RhoA (NB100-91273, NovusBiological, Milan, Italy), VHL-2 (VHL (FL-181): sc-5575, Santa Cruz Biotechnology, INC., Heidelberg, Germany), and α-Actin (monoclonal anti-α-actin (1A), sc32251, Santa Cruz Biotechnology, Inc.). The secondary antibodies used for immunoblotting were obtained from ThermoFisher Scientific (ThermoFisher Scientific, Rodano, Italy) and signals were detected using a CCD high-resolution and high-sensitivity detection technology (ChemiDoc™ XRS+ System, Bio-Rad Laboratories Srl, Segrate, Milan, Italy).

Whole-cell lysates were prepared in radio-immunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA) on ice with an added phosphatase inhibitor cocktail (PhosSTOP, Roche, Basel, Switzerland). The protein concentrations for each sample were determined using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific) according to the manufacturer’s directions. Equal amounts of protein (20 μg) were resolved by electrophoresis on 15% sodium dodecyl sulphate (SDS) polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS-T) and incubated at room temperature for 2 h with primary antibodies against Cdc42 (1:1000, ab64533, Abcam, Cambridge, MA, USA), Rac1 (1:1000, PA1-091, Invitrogen, Carlsbad, CA, USA), and RhoA (1:2000, NB100-91273, Novus Biologicals, Littleton, CO, USA). These membranes were washed out in TBS-T and incubated with secondary antibodies diluted 1:5000 for 1 h (IgG HRP-linked, anti-rabbit, and anti-mouse antibodies, respectively; Cell Signaling Technology, Danvers, MA, USA). The results were visualized using enhanced chemiluminescent (ECL) detection reagents (Millipore, Darmstadt, Germany).