Anti p c jun ser63

TPA and SP600125 were purchased from Beyotime Biotechnology. Papain was purchased from Sigma Aldrich (St. Louis, MO, USA). DNase I was purchased from Roche. The following antibodies were used: anti-JNK1 (sc-136205, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-JNK2 (sc-271133, Santa Cruz Biotechnology), anti-S-opsin (ab229786, Abcam, Cambridge, UK), anti-M-opsin (NB110-74730, Novus, Centennial, CO, USA), anti-Rhodopsin (NB120-3267, Novus), anti-Notch1 (D6F11, Cell Signaling), anti-neurofilament (ab223343, Abcam), anti-c-Jun (60A8, Cell Signaling), anti-c-Jun (sc-74753, Santa Cruz Biotechnology), anti-p-c-Jun ser63 (54B3, Cell Signaling), anti-p-c-Jun ser73 (D47G9, Cell Signaling), anti-β-actin (A5316, Sigma Aldrich), normal mouse IgG (sc-2025, Santa Cruz Biotechnology), anti-JNK (sc-7345, Santa Cruz Biotechnology), and anti-p-JNK (81E11, Cell Signaling).

Neutrophils (2 × 10⁶ cells/mL) were activated with diverse stimuli in HBSS buffer as described in figure legends. Further, cells were collected by centrifugation at 1000 RPM for 10 min and lysed using RIPA buffer [50-mM Tris pH 7.4, 15-mM NaCl, 5-mM EGTA, 0.1% SDS, 1% Sodium deoxycholate, 1% NP-40, 10-mM sodium pyruvate, 0.5-mM sodium fluoride and protease inhibitor cocktail (Roche, Switzerland)]. Equal concentrations of proteins were separated on the 10% denaturing polyacrylamide gel and blotted onto nitrocellulose membranes. The membranes were further probed using respective primary antibodies. Anti-citrullinated histone (Abcam, Cambridge, UK), Anti-p^44/42 ERK (Thr202/tyr204), Anti-ERK, Anti-pAKT (Ser473), Anti-AKT, Anti-p–C-JUN (ser 63), and Anti-C-JUN (all from Cell Signaling Technology, Massachusetts, USA) were hybridized for 1 h at room temperature and followed by horseradish peroxidase-conjugated secondary antibodies (Thermo fisher, US) for another 1 h at room temperature. Immunoblots were developed using enhanced chemiluminescence kit (ECL) (Pierce, Thermo Fisher Scientific, MA, USA) and imaged in ImageQuant Las 4000. Images of immunoblots were processed using ImageJ software (NIH, USA) for densitometric analysis.