Rabbit anti p syk

RIPA lysis buffer (Beyotime, Shanghai, China) was utilized to obtain the total proteins. After being quantified by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA), equal proteins were loaded and electrophoresed on a polyacrylamide gel, and wet-transferred to PVDF membranes (Millipore, Bedford, MA). Then the membranes were kept in 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) at indoor temperature for 1.5 h and kept in primary antibody solution with a 1:1000 dilution ratio at 4 °C for 12 h. After being treated with secondary antibody solution with a 1:5000 dilution ratio for 1 h at ambient temperature, the membranes were observed with ECL Plus WB detection reagents (Millipore).
The primary antibodies including mouse anti-phosphorylated (p)-p38, mouse anti-p38, rabbit anti-p-extracellular regulated MAP kinase (p-ERK), rabbit anti-ERK, rabbit anti-p-c-Jun NH2-terminal (p-JNK), rabbit anti-JNK, rabbit anti-β-actin, rabbit anti-p-Syk, rabbit anti-Syk, rabbit anti-p- protein kinase C delta (PKC δ), rabbit anti-PKC δ, rabbit anti-p-IKK α/β, rabbit anti-IKK α, rabbit anti-p-P65, and rabbit anti-P65 were bought from Cell Signaling Technology (Danvers, Massachusetts, USA). The rabbit anti-Histone 3 antibody was bought from Abcam (Cambridge, Massachusetts, USA).

Total protein lysates were obtained after lysing 10⁶ cells in RIPA complete buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 2mM EDTA, 1% NP40 complemented with 0.1% SDS, 1 x EDTA-free protease inhibitor cocktail (Roche, Rotkreuz, Switzerland)). Cell extracts were passed 10 times through a 25-G syringe. Protein content was determined using the Pierce BCA Protein Assay Kit (ThermoScientific, Zug, Switzerland), according to the manufacturer’s instructions. To analyze protein expression by western blot, protein (20 mg/well) was loaded into a NuPAGE 4–12% Bis-Tris Gel (Life Technologies), subjected to SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Glattbrugg, Switzerland). The membrane was incubated with rabbit anti-IRAK1 (sc-7883, Santa Cruz Biotech, Santa Cruz, CA), rabbit anti-Akt (#9272), rabbit anti-pAkt (#4058), rabbit anti-Mek1/2 (#9122), rabbit anti-pMek1/2 (#2338), rabbit anti-Syk (#12358), rabbit anti-pSyk (#2710) or rabbit anti-β-actin (# 4967, all from Cell Signaling Technology, Allschwil, Switzerland) primary antibodies; subsequently with anti-rabbit (# 7074) or anti-mouse (# 7076) IgG HRP-linked secondary antibodies (Cell Signaling Technology). The signal was detected with the ECL Western Blotting Detection Reagents (GE Healthcare) and imaged using the LAS-3000 image reader (Fujifilm, Dielsdorf, Switzerland).