Western Blot Analysis
Total proteins were extracted from cells using radio-immune
precipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO,
USA) and quantified by Bradford assay. Next, 40 μg of protein was
separated by 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and then electro-transferred to a
nitrocellulose membrane. The membrane was blocked with 5% skimmed milk
in Tris-buffered saline-Tween 20 (TBST) buffer at room temperature for 1
h. The membrane was probed with primary antibodies: rabbit anti-CD11b
antibody (Cat. No. PA5-29633; 1 : 3000), rabbit anti-Iba-1 antibody
(Cat. No. PA5-21274; 1 : 3000), rabbit anti-iNOS antibody (Cat. No.
PA3-030A; 1 : 2000), rabbit anti-β-actin antibody (No. PA1-183; 1 :
2000) at 4 °C overnight. All the antibodies above were purchased from
Invitrogen (Carlsbad, CA, USA). Subsequently, horseradish peroxidase
(HRP)-conjugated goat anti-rabbit secondary antibody (Invitrogen,
Carlsbad, CA, USA; 1 : 4000) was added and incubated with the membranes
at room temperature for 1 h. β-actin was served as an internal
reference. Enhanced chemiluminescence detection system (ECL; Amersham
Pharmacia Biotech, Piscataway, NJ) was used to develop the protein band
and Image Pro Plus 7.0 software (Media Cybernetics, Inc., Rockville,
Maryland, USA) was used for analysis.