Rabbit anti rhoa

Thrombin (HCT-0020) was from Haematologin Technologies, Nocodazole (M1404) was from Sigma, recombinant human TNFα was from R&D systems and 8-pCPT-2-O-Me-cAMP-AM (007) was from Tocris Bioscience. Monoclonal antibodies (mAb) Rabbit anti-RhoA and Rabbit anti-RhoC were from Cell Signaling. Polyclonal antibody (pAb) Rabbit anti-RhoB was from Santa Cruz Biotechnology. Actin-stain 555 Phalloidin was obtained from Cytoskeleton. mAb Mouse anti-VE-cadherin/CD144 AF647 was from BD Pharmingen. mAb Mouse anti-actin for immunoblotting was from Sigma. Secondary antibody Chicken anti-Rabbit labeled with Alexa488 for immunofluorescence was from Invitrogen. Secondary HRP-labeled antibodies Goat anti-Mouse and Swine anti-Rabbit for immunoblotting were purchased from Dako.

Rat cortical neurons plated in 10 cm² dishes (approximately 10 million neurons per dish) were used with a commercially available Rho Activation Assay Kit (Millipore). The neurons were placed in plain NB media for 4 h prior to assay. In brief, following the manufacturers protocol, we lysed the cortical neurons on ice with MLB buffer supplemented with anti-protease and anti-phosphatase cocktails (Calbiochem) and spun the lysates at 14 000 × g for 5 min. The supernatant was collected and added to 35 µl of agarose beads coupled to Rhotekin Rho Binding Domain and rocked in the cold room for 45 min, a small sample of each supernatant was not added to the beads and was used as total Rho loading controls. Beads were washed three times with MLB buffer and 20 µl 2× Laemmeli buffer was added and samples were prepared for Western blotting. Proteins were separated on pre-cast 4–20% gradient gels (Thermo Fisher Scientific) and transferred to nitrocellulose at 75 V for 1 h. Membranes were successfully probed with rabbit anti-RhoA (1:1000; Cell Signaling Technology) and HRP conjugated anti-rabbit IgG (1:2000; Cell Signaling Technology). Membranes were reacted with Pierce ECL Western Blotting Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Densitometric measurements were made using NIH ImageJ software.

Primary antibodies used were rabbit anti-Myc (Cell Signaling Technology, 2272, used at 1:1000 for western blots), mouse anti-Myc (Cell Signaling Technology, 2276, used at 5 μg/ml for co-immunoprecipitation), rabbit anti-FLAG (Cell Signaling Technology, 2368, used for co-immunoprecipitation experiments involving C3 transferase at 1:1000), rabbit anti-GFP (Cell Signaling Technology, 2555, used for co-immunoprecipitation experiments involving C3 transferase at 1:1000), rabbit anti-GFP (Thermo Fisher Scientific, A-11122, used at 1:1000 for western blot), phospho-ERK1/2 (Cell Signaling Technology, 9101, used at 1:1000 for western blot), rabbit anti-RhoA (Cell Signaling Technology, 2117, used at 1:1000 for western blot), rabbit anti-RhoB (Cell Signaling Technology, 2098, used at 1:1000 for western blot), rabbit anti-RhoC (Cell Signaling Technology, 3430, used at 1:1000 for western blot) Secondary antibodies used were HRP-conjugated monoclonal mouse anti-rabbit IgG, light chain specific (Jackson ImmuonoResearch, 211-032-171, clone 5A6-1D10, used at 1:100,000) and goat anti-rabbit IgG (Li-COR, 926-32211, used at 1:50,000).