Anti rhob

Confluent monolayers of HUVECs grown on fibronectin-coated 55 cm² dishes were treated as indicated. Additionally, all samples were treated with 5 μM MG132 (#S2619, Selleckchem) for 2 h. Cells were washed with PBS supplemented with 1 mM CaCl₂ and 0.5 mM MgCl₂ and lysed in 400 μl ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 1× protease inhibitor cocktail). Lysates were centrifuged at 14000 rpm and 4 °C for 10 min, and 8% of supernatant was taken as input and mixed with 3× SDS sample buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 100 mM DTT, 0.02% Bromophenol Blue). The remaining supernatant was incubated with 1 μg anti-RhoB (#sc-8048, Santa Cruz Biotechnology) and rotated overnight at 4 °C. Twenty-five microliter Dynabeads Protein G (#10003D, Thermo Fisher Scientific) were added to the lysate and incubated for 1 h rotating at 4 °C. The beads were washed four times with lysis buffer and ultimately lysed in 30 μl 2× SDS samples buffer. The input and IP samples were analyzed by Western blot.

Cells were harvested in 1% Triton X-100 lysis buffer (150 mM NaCl, 20 mM Tris HCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 1% deoxycholate, at pH 7.4). All lysis buffers were supplemented with 1 mM PMSF, 1 mM sodium vanadate, 1 mM sodium fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Samples were resolved by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with 5% BSA and probed as described with appropriate antibodies: anti-Arf6 (sc-7971), anti-RhoA (sc-418), anti-RhoB (sc-8048), anti-RhoC (12116), and GST.B-14 (Santa Cruz Biotechnology); HA 0.11 (Berkeley); GFP (Roche); and anti-β-actin (AC-15; Sigma-Aldrich). This was followed by incubation with HRP-conjugated secondary antibodies. All immunoblots were visualized by enhanced chemiluminescence (Amersham Biosciences). For immunoprecipitations, lysates were incubated with antibody at 4°C overnight with gentle rotation followed by 1-h incubation with protein A or G Sepharose beads. Captured proteins were collected by washing three times in lysis buffers, eluted by boiling in SDS sample buffer, and processed as above for Western blotting.