Mouse anti jnk

The C7 dorsal spinal cord tissues OR BV-2 cells were processed as described in our previous study [40 (link)]. Briefly, after centrifugation at 12,000 rpm at 4^oC for 15 min, the supernatants were collected. Protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific, USA). The protein lysates were separated using 10% SDS-PAGE, then electrophoretically transferred onto the PVDF membranes (Roche Applied Science, Germany). After blocking with 5% nonfat milk in TBS-T for 1 h at RT, membranes were incubated with the specific antibodies: mouse anti-protein kinase C (PKC, 1:500, Santa Cruz), rabbit anti-phospho-extracellular signal-regulated kinase (p-ERK, 1:1000, Cell Signaling Technology), rabbit anti-ERK (1:1000, Cell Signaling Technology), mouse anti-phospho-c-jun (p-c-jun, 1:500, Santa Cruz), mouse anti-c-jun (1:500, Santa Cruz), mouse anti-phospho-c-jun N-terminal kinase (p-JNK, 1:500, Santa Cruz), mouse anti-JNK (1:500, Santa Cruz), mouse anti-gp91phox (1:500, Santa Cruz), mouse anti-p22phox (1:5000, Santa Cruz), rabbit anti-p47phox (1:500, ABclonal), mouse anti-p67phox (1:500, Santa Cruz) and rabbit anti-GAPDH (1:10000, Abcam) for overnight at 4°C, and then incubated with the secondary antibodies for 1 h at room temperature. The protein bands were quantified by Image J software, using GAPDH as the internal control.

The RAW 264.7 macrophage cell line was obtained from ATCC (Manassas, VA, USA). Cells were incubated in Dulbecco’s modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; GIBCO BRL, Grand Island, NY, USA) at 37 °C in a humidified incubator under 5% CO₂. L7Gn was purchased from NPBANK (National Institute for Korean Medicine Development, Gyeongsan, Korea). LPS and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). EZ-Cytox solution was from DAEIL lab (Seoul, Korea). TOPscript cDNA synthesis kit and 2X TOPsimple™ DyeMIX-nTaq were purchased from Enzynomics (Daejeon, Korea). Rabbit anti-inhibitor of κBα (IκBα), rabbit anti-phospho (p)-IκBα (Ser-32/36), mouse anti-JNK, mouse anti-p38, rabbit anti-iNOS, goat anti-COX-2, rabbit anti-IL-1 receptor-associated kinase 1 (IRAK1), mouse anti-α-tubulin, and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p-p38 (Thr-180/Tyr-182), mouse anti-p-ERK (Thr-202/Tyr-204), rabbit anti-ERK, rabbit anti-p-JNK (Thr-183/Tyr-185), rabbit anti-transforming growth factor-β-activated kinase 1 (TAK1), and rabbit anti-p-TAK1 (Thr-184/Tyr-187) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).