Rabbit α rhob antibody

Immunoprecipitation of endogenous RhoB was performed with rabbit α-RhoB antibody (Santa Cruz Biotechnology) from confluent 60 cm² dishes of primary HUVECs transfected with control siRNA or FBXW7 siRNA. Proteasomal degradation was inhibited by adding 5 µM MG132 at 2 h before lysis. Upon stimulation, cells were washed in phosphate-buffered saline (PBS) containing 1 mM CaCl₂ and 0.5 mM MgCl₂ and lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, complete protease inhibitor cocktail tablets [Roche]) and phosphatase inhibitors (1 mM Na₃VO₄ and 25 mM NaF). Lysates were cleared by centrifugation and incubated with 1 µg RhoB antibody for 2 h at 4°C. RhoB-containing complexes were pulled out by incubation with Dynabeads protein G (Thermo scientific) for 1 h at 4°C. Finally, beads were washed four times with lysis buffer, and immunoprecipitated proteins were eluted with sample buffer and analyzed with SDS–PAGE.

Immunoprecipitation of endogenous RhoB was performed with rabbit αRhoB antibody (Santa Cruz) from confluent 60-cm² dishes. Before lysis, cells were stimulated with either 10 ng/ml TNF-α (Peprotech) or 300 nm MLN4924 for 4 h or 2.5 µM PR619 for 2 h. Proteasomal degradation was inhibited by adding 5 µM MG132 at 2 h before lysis. Upon stimulation, cells were washed in PBS containing 1 mM CaCl₂ and 0.5 mM MgCl₂ and lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% NP-40), cOmplete Protease Inhibitor Cocktail Tablets (Roche), and phosphatase inhibitors (1 mM Na₃VO₄ and 25 mM NaF). Lysates were cleared by centrifugation and incubated with 1 µg RhoB antibody for 2 h at 4°C. RhoB containing complexes were pulled out by incubation with Dynabeads protein G (Thermo Fisher Scientific) for 1 h at 4°C. Finally, beads were washed four times with lysis buffer, and immunoprecipitated proteins were eluted with sample buffer and analyzed with SDS-PAGE.