Rabbit anti erk1 2

Immunoblotting was done as previously described (15 (link)) employing the following antibodies and dilutions: anti-NS3 antibodies were described in Ref. (48 (link)), rabbit anti-phospho-Tyr701-STAT1 (cat. #9167; 1:1,000), rabbit anti-STAT1 (cat. #9172; 1:1,000), rabbit anti-phospho-Tyr690-STAT2 (cat. #88410; 1:1,000), rabbit anti-STAT2 (cat. #72604; 1:1,000) rabbit anti-phospho-Tyr705-Stat3 (cat. #9145; 1:1,000), mouse anti-STAT3 (cat. #9139; 1:1,000), and rabbit anti-SOCS3 (cat. #2923; 1:1,000), (all from cell signaling); mouse anti-phospho-ERK1/2 (Sigma-Aldrich, cat. #M8159; 1:500), rabbit anti-ERK1/2 (Santa-Cruz Biotechnology, cat. #sc-154; 1:10,000), rabbit-anti-JAK1 (Santa-Cruz Biotechnology, cat. #sc-277; 1:400), mouse anti-Actin (MP Biomedicals, cat. #69100; 1:10,000), rabbit anti-GAPDH (Abcam, cat. #ab8245; 1:5,000), mouse anti-GFP (MBL, cat. #M048-3; 1:1,000), and HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, cat. #115035003; 1:15,000).

The tissues or cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mMNaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease inhibitors (Roche Applied Science). The lysates were clarified by centrifugation at 13,000 × g at 4 °C for 20 min, and the supernatants were collected and normalized for protein concentration. Proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking with PBS containing 5% skim milk and 0.05% Tween 20, the membranes were incubated with primary antibodies.For detection, a fluorescence-conjugated secondary antibody and an electrogenerated chemiluminescence system (GE Healthcare) were used. The membrane was exposed to an imaging system (LAS-3000, Fujifilm) according to the manufacturer’s specifications. The protein bands were quantified using ImageJ 1.44p software. The following antibodies were used: rabbit anti-p-ERK1/2 (1:1000,Santa Cruz, sc-7383); rabbit anti-ERK1/2 (1:1000,Santa Cruz,sc-292838), rabbit anti-GAPDH (1:5000, Abcam, ab128915). Horseradish peroxidase-conjugated rabbit IgG-specific (1:5000, Cell Signaling Technology, 7074 S) were used for secondary antibodies. Full-length gel is available at Supplementary data.

Lipopolysaccharide derived from Escherichia coli O111:B4, TRAP-6, ADP and prostaglandin (PG) E1 were obtained from Sigma-Aldrich (St. Louis, MO, United States). Pam3CSK4 was purchased from InvivoGen (San Diego, CA, United States) and thrombin from Biopool (Umea, Sweden). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human P-selectin (CD62P), anti-CD40L (CD154), and CD63 and PE-conjugated mouse anti-human CD41, TLR2, and TLR4 were from BD Biosciences (San Jose, CA, United States). FITC-conjugated CD45 and Human CCL5 (RANTES) ELISA Max were purchased from BioLegend (San Diego, CA, United States) and Quantikine ELISA Human P-selectin/CD62P was from R&D Systems (Minneapolis, MN, United States). Rabbit anti-human VWF and HRP-conjugated anti-human VWF were from Dako (Glostrup, Denmark), mouse anti- p-ERK1/2 (Tyr 204) and rabbit anti ERK 1/2 were obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Cell protein lysate was obtained using cell extraction buffer (Life Technologies, Carlsbad, CA, USA) followed by incubation for 30 min, at 4 °C, on a shaker. After centrifugation, the supernatant was submitted to protein quantification by a BCA Protein Assay Kit. SDS-Page and Western blots were performed as described using stain-free gels [28 (link)]. Membranes were incubated with mouse anti-GAPDH (GeneTex, Irvine, USA), rabbit anti-pERK 1/2, rabbit anti-Trx1 (Cell Signaling Technology, Frankfurt am Main, Germany), rabbit anti-ERK 1/2, rabbit anti-Nrf2, mouse anti-α-Tubulin (Santa Cruz Biotechnology, Heidelberg, Germany) as well as goat anti-rabbit and anti-mouse IgG kappa binding protein IgG HRP-conjugated (Santa Cruz Biotechnology) diluted in 1% BSA solution. Western blots were quantified via chemiluminescence using a Clarity Western ECL substrate chemiluminescence kit (BioRad Laboratories GmbH, Munich, Germany). Besides the loading controls GAPDH and α-tubulin, we also used stainfree total protein normalization. Membranes were photographed and analyzed using a ChemiDoc XRS + imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad).

The protocols of the Western blot were performed as described by Hanson et al. [32 (link)]. Briefly, protein extracts, quantified by a Bradford Protein Assay (Bio-Rad Laboratories, CA, USA), underwent SDS-polyacrylamide gel electrophoresis and were transferred to Immobilon-P membranes. The following antibodies were used: rabbit anti-ERK1/2, goat anti-Matrin3, goat anti-βactin, anti-p22phox (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 500, rabbit anti-Akt1, rabbit anti-Rac1, and rabbit anti-ERK1/2 (Cell Signalling Technology, Beverly, MA, USA), mouse anti-tubulin, rabbit anti-Nox1, and mouse anti-sc-35 (Sigma Aldrich St. Louis, MO, USA), rabbit anti-Nox4 (Novus Biologicals, CO, USA), rabbit anti-Nox2, and mouse anti-pH2A(Ser139) (Millipore, Billerica, MA, USA) diluted 1 : 1000; peroxidase-labelled anti-rabbit, mouse and goat secondary antibodies diluted 1 : 3000 (Pierce Antibodies, Thermo Scientific; Rockford, IL, USA). Ab dilution was performed in TBS-T pH 7.6 containing 3% BSA. The membranes were visualized using Supersignal substrate chemiluminescence detection kit (Pierce, Rockford, IL, USA). Anti-βactin antibody was used as control of protein loading. Quantization of the signal was obtained by chemiluminescence detection on a Kodak Image Station 440CF and analysis with the Kodak 1D Image software.