Tonitrocellulose membranes

After treatment with the indicated inhibitors with or without
thrombin-mediated activation, platelet whole cell lysates were generated in TNES
buffer (50mM Tris pH7.5, 100mM NaCl, 2mM EDTA, 1% NP-40 supplemented with
protease and phosphatase inhibitors (cOmplete and PhosSTOP cocktail tablets,
respectively, Sigma-Aldrich, St. Louis, MO, USA). After cellular debris was
removed via high speed centrifugation, equal amounts of the lysates were
fractionated on gradient 4–20% SDS-PAGE gels and transferred to
nitrocellulose membranes (both from Bio-Rad, Hercules, CA, USA). The membranes
were analyzed for immunoreactivity to the following antibodies: Hsp40, (Cell
Signaling Technology, Danvers, MA, USA), Hsp70, Hsp90, and Grp94 (Enzo Life
Sciences, Farmingdale, NY, USA), and α-tubulin (EMD Millipore,
Burlington, MA, USA). Bound antibodies were detected via species-specific
horseradish peroxidase (HRP)- conjugated secondary antibodies at a 1:000
dilution (GE Healthcare, Pittsburgh, PA, USA), followed by reaction with Pico
ECL (Thermo Scientific, Rockford, IL, USA) to produce a chemiluminescent signal,
and exposed to x-ray film (Vita Scientific, College Park, MD, USA).

Cells growing in monolayers were lysed using Cell Extraction Buffer (Life
Technologies) supplemented with complete protease inhibitors and PhosSTOP phosphatase
inhibitor cocktail tablets (Roche). Cell lysates were cleared by centrifugation, protein
concentrations were determined by DC Protein Assay (BioRad), and denatured lysates were
run on 4–12% Bis-Tris gradient gels (Invitrogen). Gels were transferred to
nitrocellulose membranes (BioRad) before being immunoblotted with indicated antibodies.
Cleaved-PARP antibody was obtained from Cell Signaling Technology. RAC1 activation assays
were performed as previously described according to the manufacturer's protocol (Cell
Biolabs) (10 (link)).