Protease and phosphatase inhibitor tablet

Neurons were harvested in ice-cold lysis buffer (150 mM NaCl, 20 mM Tris, 5 mM EDTA, 10% glycerol, 1% Igepal, protease and phosphatase inhibitor tablets (Roche)), lysed for 30 min on ice and cleared by centrifugation at 15.000 rpm for 15 min at 4C. Protein concentrations were determined using Roti-Quant Universal kit (Roth). Protein samples were loaded on 7.5% or 10% polyacrylamide gels for SDS-PAGE. Following transfer to 0.2 µm pore-size PVDF membranes, blots were blocked in 5% BSA in TBST for 1 hr at RT. Blots were then probed with antibodies against VEGFR2, (phospho-) VEGFR2 (Y1175), GFAP and VE-Cadherin. After incubation with appropriate HRP-conjugated secondary antibodies, bands were detected with ECL Western Blotting detection reagent (Millipore) and visualized using ImageQuant LAS 4000 (GE Healthcare).

Whole cell extracts were prepared by lysing cells in RIPA buffer (100 mM Tris-HCl pH 8.0, 140 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, protease and phosphatase inhibitor tablets (Roche)) on ice. Whole cell extracts were collected after centrifugation at 20,000 g for 15 min at 4° C. Proteins were quantified by Bradford assay and immunoblot analysis was performed as described previously (Farabaugh et al., 2017 (link)). Densitometry analysis was performed using Image J.

RAW 264.7 cells transfected with siRNA directed against one of the three RhoGAP candidates or with a non-targeting siRNA control were washed with PBS and lysed with cold RIPA buffer (Sigma-Aldrich) supplemented with protease- and phosphatase-inhibitor tablets (Roche). 40 μg of total protein were loaded per lane of a 12% SDS–polyacrylamide gel electrophoresis and separated by electrophoresis, before being transferred to methanol-activated polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 and 5% bovine serum albumin for 30 min. This solution was also used to dilute the primary anti-RhoGAP and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies, which were incubated for 2 h and 45 min, respectively. Primary antibodies against ARHGAP12, ARHGAP25 and SH3BP1 were used at 1:250, 1:1,000 and 1:1,000 dilutions, respectively. HRP-conjugated antibodies were used at a 1:10,000 dilution. Membranes exposed to ECL western blotting substrate (GE Life Sciences) were visualized using the Odyssey Fc (LI-COR) system. Brightness/contrast parameters were adjusted globally across the entire image using the LI-COR Image Studio software. Immunoblots were cropped for presentation; see Supplementary Fig. 6 for uncropped images.