Anti c jun rabbit antibody

PD098059, LY294002, SP600125 and SB203580 chemical inihbitors were obtained from Sigma (St Louis, MO). Calcein-AM was obtained from Invitrogen Molecular Probes (Burlington, ON). Dimethylsulfoxyde was purchased from Fisher (Montreal, QC). IL-1β was obtained from R&D Systems (Minneapolis, MN). Anti-phospho-ERK1/2 MAPK (T²⁰²/Y²⁰⁴) mouse antibody, anti-phospho-Akt (S⁴⁷³) rabbit antibody, anti-Akt rabbit antibody, anti-phospho-c-Jun (S⁶³) rabbit antibody, anti-c-Jun rabbit antibody were obtained from Cell Signaling Technology (Beverly, MA). Anti-E-selectin mouse monoclonal antibody was obtained from R&D Systems (Minneapolis, MN). Anti-GAPDH mouse antibody was obtained from Novus Biologicals (Oakville, ON). Anti-actin rabbit antibody was obtained from Sigma (St Louis, MO). Anti-ERK1/2 rabbit antibody, anti-phospho-HSP27 rabbit antibody, anti-HSP27 rabbit antibody were kind gifts from Dr. Jacques Landry (CRCHU de Québec-Université Laval, QC). Anti-mouse/rabbit-IgG-horseradish-peroxidase (HRP) goat antibodies were obtained from The Jackson Laboratory (Bar Harbor, ME).

Cells were lysed using SDS-PAGE loading buffer without reducing agents. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Antibodies were applied according to their manufacturers’ protocols. Blots were developed with SuperSignal West Pico Substrate (Thermo Fisher Scientific, Montreal, QC). Anti-phospho-ERK1/2 MAPK (T²⁰²/Y²⁰⁴) mouse antibody (1:1000), anti-ERK1/2 MAPK mouse antibody (1:1000), anti-phospho-Akt (S⁴⁷³) rabbit antibody (1:1000), anti-Akt rabbit antibody (1:1000), anti-phospho-c-Jun (S⁶³) rabbit antibody (1:1000), anti-c-Jun rabbit antibody (1:1000), anti-phospho-p65 (S⁵³⁶) rabbit antibody (1:1000), anti-p65 rabbit antibody (1:1000), anti-phospho-HSP27 (S⁸²) rabbit antibody (1:1000) and anti-HSP27 rabbit antibody (1:1000) were obtained from Cell Signaling Technology (Beverly, MA), Anti-E-selectin mouse monoclonal antibody (1:1000) was obtained from R&D Systems (Minneapolis, MN). Anti-METTL3 rabbit antibody (1:2000) was obtained from Abcam (Cambridge, UK). Anti-GAPDH mouse antibody (1:10000) was obtained from Novus Biologicals (Oakville, ON). Anti-mouse/rabbit-IgG-horseradish-peroxidase (HRP) goat antibodies (1:5000) were obtained from The Jackson Laboratory (Bar Harbor, ME).

Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. Ten μg of proteins were electrophoresed on 10% or 15% SDS-PAGE, transferred onto PVDF membrane, blocked with 5% BSA, and then incubated with a rat anti-FABP5 antibody (Clone # 311215, R&D systems), a rabbit anti-c-Jun antibody (Cell signaling), a rabbit anti-phosphoSer63-c-Jun antibody (Cell signaling), or a rabbit anti-phosphoSer73-c-Jun antibody (Cell Signaling) overnight at 4°C. After washes in PBS with 0.1% Tween-20, the membranes were incubated with anti-rat or anti-rabbit IgG conjugated to horseradish peroxidase for FABP5, c-Jun and phospho-c-Jun protein detection. Membranes were stripped and re-probed with a mouse anti-GAPDH antibody (6C5 sc-32233, Santa Cruz Biotechnologies). Densitometry was performed to quantify proteins of interest.