Protease inhibitor cocktail

Immunoblot experiments were conducted by following the protocol described in Cáceres et al. (2015) (link). Briefly, transfected HEK293 cells were washed once with ice-cold DPBS and lysed with 150 μL of ice-cold lysis buffer containing 1% (v/v) Triton X-100 (Merck, 108603), 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 1 mM sodium orthovanadate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma, 78830), and protease inhibitor cocktail (Cytoskeleton, Inc., PIC02) for 10 min at 4°C. The lysates were centrifuged at 12,000 g at 4°C for 10 min. The supernatants were mixed with 150 μL of 2x Reducing Sample Buffer [RSB: 60 mM Tris-HCl pH 6.8, 25% (v/v) glycerol, 2% (w/v) SDS (Sigma, L5750), 14.4 mM 2-mercaptoethanol, 0.1% (w/v) bromophenol blue] and size-fractionated by 7.5% SDS-PAGE. Lauryl sulfate (Sigma, L5750) was the form of SDS used in all gel solutions. The immunoblots were visualized by Pierce ECL Western Blotting Substrate (ThermoFisher, 34080) and images were acquired with a MiniHD9 imager (Uvitec).

All steps of the microtubule sedimentation assay were performed at 37°C to maintain the microtubule network of the samples. Samples were lysed at 37°C for 10 min in warm 1:1 Lysis and Microtubule Stabilization Buffer (Cytoskeleton Inc., Denver, CO) and water, supplemented with 20μM taxol, 1mM ATP, 100μM GTP, and 1% protease inhibitor cocktail (Cytoskeleton Inc., Denver, CO). The lysate was syringe sheared ten times and spun at 2000 x g at 37°C for 5 min to clear out debris. The pre-cleared supernatant was then subjected to ultracentrifugation at 100,000 x g for 1 hour at 37°C to separate the soluble fraction and the microtubule pellet. These fractions were denatured and analyzed for relevant proteins using SDS-PAGE.

pGST-Rhotekin-RBD and pGST-PAK1-PBD plasmids (Addgene, Watertown, MA) were separately transformed into bacteria to produce recombinant fusion proteins. Afterwards, glutathione Sepharose 4B resin (GE Healthcare Life Sciences, Pittsburg, PA) was added to the bacterial lysate for centrifugation. Then, the pellet was re-suspended in PBS with 10% glycerol, stored at − 80 °C in 20-μl aliquot, and used as baits later for active RhoA and active Rac1 pull-down assay, respectively^{51 (link)}. Before pull-down, the aliquot was quantified using BSA as standard for consistency. Cell lysates were prepared in RIPA lysis buffer (10-mM Tris HCl, pH 7.4, 150-mM NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) SDS, 0.5% (v/v) DOC and 1-mM EDTA), containing protease inhibitor cocktail (Cytoskeleton, Inc., Denver, CO), and cleared by centrifugation (20,000 × g). Then, each lysate sample was mixed with an equal amount of pull-down resin and incubated at 4 °C overnight. The next day, the sample was pulled down by centrifugation, washed 3 times by lysis buffer before the resin-bonded protein was dissociated by 2 × SDS sample buffer for 2 min at room temperature. The sample was then boiled for 5 min, and subjected to 12% SDS PAGE, following by Western blotting.

Platelets (2 × 10⁸/ml) were allowed to spread on 10 cm dishes coated with 100 µg/ml fibrinogen for 25 minutes before washing and treatment with either vehicle or S-nitrosoglutothione (1 µM) ± 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (2 µM) for 20 minutes. Adhered platelets were lysed in cell lysis buffer supplemented with protease inhibitor cocktail (Cytoskeleton, Denver, USA) and frozen. Protein quantitation was performed via Precision Red reagent (Cytoskeleton, Denver, USA) with a 96-well plate reader. 50 µg of rhotekin-RBD beads (Cytoskeleton, Denver, USA) were loaded with 200 µg of protein and agitated for 90 minutes at 4 °C. Samples were processed as per kit instructions. Briefly, pull down beads were centrifuged at 4500 × g at 4 °C for 1 minute and washed with wash buffer before being pelleted out again at 4500 × g for a further 3 minutes. Wash supernatant was discarded and the beads were resuspended in 2× laemmli buffer and ran through SDS-PAGE/immunoblot with respective total RhoA controls and probed with anti-RhoA antibody.

RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction. In brief, cells were lysed in lysis buffer [50 mM Tris pH 7.5, 10 mM MgCl₂, 0.5 mM NaCl, 2% NP-40, protease inhibitor cocktail (Cytoskeleton, Inc)] and the same amount (300–400 µg) of cell lysates was incubated with either 50 µg of PBD-GST or RBD-GST protein beads to precipitate activated Rac1 and Cdc42 or RhoA. Active (GTP-bound) and total Rac1, Cdc42, and RhoA were assessed by immunoblotting with anti-Rac1 (BD Bioscience), anti-Cdc42 (BD Bioscience), and anti-RhoA (Santa Cruz) antibodies. Relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis with image J (NIH) software. Experiments were repeated at least 4–5 times and the data was expressed as means ± S.E.M. ANOVA was used for multiple-group comparisons and the unpaired Student’s t test was used for two-group comparisons. Differences were considered statistically significant when P<0.05.

An actin polymerization assay was performed according to the protocol described previously.³⁰ Each aortic strip was placed in a 0.5 mL tissue homogenization tube (Precellys, Bertin Technologies) containing pre‐warmed lysis and F‐actin stabilization buffer (in mmol/L: 50 PIPES pH 6.9, 50 NaCl, 5 MgCl₂, 5 EGTA) 5% [V/V] Glycerol, 0.1% Nonidet P40, 0.1% Triton X‐100, 0.0% Tween 20, 0.1% 2‐mercapto‐ethanol, 0.001% Antifoam C with 1 mmol/L ATP and 1× protease inhibitor cocktail (Cytoskeleton, Inc). The tissue was quickly homogenized. 100 μL of homogenates was incubated at 37°C for 20 minutes and spun at 150 000 g for 60 minutes at 37°C in Optima TLX ultracentrifuge (Beckman Coulter). The supernatant (G‐actin) was carefully pipetted, 100 μL of ice‐cold F‐actin depolymerization buffer (8 mol/L urea) was added to each pellet, and the pellet was dissolved by triturating every 15 minutes for 1 hour. The solution was spun at 2300 g for 5 minutes at 4°C, and the supernatant collected to give the final F‐actin fraction. Equal volume of G and F‐actin samples was resolved by SDS‐PAGE and immunoblotting with anti‐actin primary antibody (Cytoskeleton).