Tbs t

Cells were harvested and total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,.1% sodium dodecyl sulfate, 1 mM PMSF) supplemented with protease inhibitor cocktail (Cell Signaling Technology, Massachusetts, USA). The protein concentration was measured using bradford protein assay (Sigma, Missouri, USA). The total protein extract was resolved on SDS-PAGE and then transferred onto either 0.22 μm or 0.45 μm PVDF membrane (Millipore, Massachusetts, USA). After the transfer, using protein ladder as reference, the membranes were cut according to the protein of interest as shown in the results (also refer to the raw images provided in the supplementary), then blocked with either 5% BSA (SRL, Mumbai, India) or 5% fat-free milk in TBST (Amersham, GE, Chicago, USA) for 1 hour at 4 °C followed by 1 hour at room temperature with gentle shaking. The membrane was then washed and probed with primary antibody overnight as per manufacturers’ instructions followed by washing and then incubating with secondary antibody (in TBST with 2.5% fat-free milk). The blot was then developed by enhanced chemiluminescence (ECL) substrate (Bio-Rad, California, USA) and documented using the chemidoc (Bio-Rad XRS+).

Platelets lysed in Laemmli buffer were separated by SDS PAGE gel electrophoresis and probed for eIF-4E, vinculin and β-actin. After protein transfer to a nitrocellulose membrane, membranes were blocked in 5% non-fat milk in Tris-buffered saline with Tween-20 (TBS-T, Sigma-Aldrich, Taufkirchen, Germany) for 1 h. Afterwards, primary monoclonal antibodies were added. eIF4E, actin and vinculin were detected using specific mouse anti-human antibodies (1:1000) (anti-eIF4E, BD Transduction Laboratories, San Diego, CA, USA; anti-actin, ICN Biomedicals, Costa Mesa, CA, USA; anti-vinculin, Upstate Biotechnology, Placid, NY, USA) and incubated overnight at 4 °C with constant agitation. Membranes were washed in TBS-T repeatedly, and HRP-conjugated secondary antibody (1:10,000) (GE Healthcare, Freiburg, Germany) was added for 1 h at room temperature. After washing, chemiluminescent substrate (ECL reagent, Amersham Biosciences, Munich, Germany) was added for 1–5 min and bands were visualized on plain film. β-Actin served as loading control (1:1000, rabbit anti-human β-Actin, Cell Signaling Technology, Dallas, TX, USA).

Platelets lysed in Laemmli buffer were separated by SDS PAGE gel electrophoresis and probed for HSP27, phospho-HSP27, and β-actin. After protein transfer to a nitrocellulose membrane, membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween-20 (TBS-T, Sigma-Aldrich, Taufkirchen, Germany) for 1 h. Afterwards, primary monoclonal antibody to HSP27 or phospho-HSP27 (1:1000, Santa Cruz Biotechnologies, Dallas, TX, USA) were added and incubated overnight at 4 °C with constant agitation. Membranes were washed in TBS-T repeatedly, and HRP-conjugated secondary antibody (1:10000) (GE Healthcare, Freiburg, Germany) was added for 1 h at room temperature. After washing, chemiluminescent substrate (ECL reagent, Amersham Biosciences, Munich, Germany) was added for 1–5 min and bands were visualized on plain film. β-Actin served as loading control (1:000, rabbit anti-human β-Actin, Cell Signaling Technology, Dallas, TX, USA). HSP27 and phospho-HSP27 levels were quantified by band densitometry analysis with the help of HSP27- and pHSP27-to-actin ratios. Mean HSP27- and pHSP27-to-actin ratios for the myocardial infarction group were set as 1 and relative increases in HSP27 and pHSP27 levels in the control group were compared accordingly. Relative HSP27 and pHSP27 levels between groups based on actin loading are shown.