Anti p47phox antibody

Phorbol ester phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLF), C5a and isoluminol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The p38 MAPK inhibitor SB203580 was ordered from Selleck (Houston, TX, USA). Antibodies including Phosphor-Akt, Akt, Phosphor-p38 MAPK, p38 MAPK, MK2, and GAPDH were ordered from Cell Signaling Technology (Danvers, MA, USA). Anti-p47^phox antibody was purchased from Santa Cruz Biotechnology and anti-phospho-p47^phox (Ser329) antibody was generated by N.J. Compass Biotechnology (Nanjing, China). Other reagents were ordered from Sigma-Aldrich (St. Louis, MO, USA).

Thoracic aorta sections (5 μm) were sliced from paraffin blocks fixed with 4% paraformaldehyde. Antigen retrieval was performed by heating the sections in a 60 °C oven with a 10 mM citrate buffer overnight. After cooling, the sections were incubated with 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidases. A rabbit polyclonal anti-p22phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:50 dilution for p22phox immunostaining. An Alexa Fluor 488-conjugated secondary antibody (Invitrogen Corp., Carlsbad, CA, USA) was used at a 1:2000 dilution. For p47phox immunostaining, the sections were incubated with a rabbit polyclonal anti-p47phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted at 1:50 in 0.5 M TBST at 4 °C overnight. An Alexa Fluor 555-conjugated secondary antibody (Invitrogen Corp., Carlsbad, CA, USA) was used at a 1:2000 dilution. Nuclear counterstaining was accomplished with DAPI at a 1:10,000 dilution. The sections were stored in the dark until they were analyzed using a confocal microscope (LSM 510 META, Carl Zeiss, Inc., Overkochen, Germany).

BMDM were lysed in cell lysis buffer (9803, Cell Signaling Technology, Danvers, MA, USA) with protease inhibitor cocktails and PMSF (P8340 and 93482, Sigma-Aldrich). The lysed cell protein was immunoprecipitated with anti-p47^phox antibody (sc-14015, Santa Cruz Biotechnology) using Dynabeads protein G immunoprecipitation kit (10007D, Thermo Fisher Scientific). The immunoprecipitated proteins were then subjected to immunoblotting analysis using anti-phosphoserine antibody (61–8100, Thermo Fisher Scientific) and anti-gp91^phox antibody (sc-5827, Santa Cruz Biotechnology), respectively.