Mouse anti p22phox

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.

Microglia (1 × 10⁵) were plated on poly-l-lysine glass coverslips (Fisher Scientific, Pittsburgh, PA, 08-774-383) in 12-well plates. Cells were treated for 18 h with vehicle (media) or 100 ng/mL LPS in media supplemented with 2% FBS (2 mL solution per well; Hyclone, GE Healthcare Life Sciences, Marlborough, MA). Immunocytochemistry was performed via conventional techniques. That is, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% Triton X-100. Blocking was performed with 10% normal donkey serum (Jackson Laboratories, Bar Harbor, ME) for 3 h before being incubated with primary antibodies. Goat anti-TSPO (Abcam, Cambridge, MA, ab118913, RRID:AB_10898989, 1:500); mouse anti-gp91^phox (BD Biosciences, San Jose, CA, 611415, RRID:AB_398937, 1:100); mouse anti-p22^phox (Santa Cruz Biotechnology, Dallas, TX, sc-130,551, RRID:AB_2245805, 1:100); rabbit anti-VDAC (Abcam, Cambridge, MA, ab15895, RRID:AB_2214787, 1:500) were used. After washing, cells were incubated with appropriate Alexa Fluor secondary antibodies for 1 h (Life Technologies, Carlsbad, CA; AF 488, 594, and 647, 1:500). Coverslips were mounted with Prolong with DAPI (Life Technologies, Carlsbad, CA) to counterstain for cell nuclei.