The lysed bPAEC were assayed for arginase I and arginase II protein by
Western blot analysis as previously described (18 (link), 26 (link)). Aliquots of cell
lysate (15-20 μg of protein) were diluted 1:1 with SDS sample buffer,
heated to 95°C for 5 minutes, and electrophoresed on polyacrylamide gel.
Separated proteins were electrotransferred to PVDF membranes, incubated with
5% nonfat milk for one hour, and then incubated overnight with primary
antibody, arginase I (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or
arginase II (1:500; Santa Cruz) overnight and then washed three times with
PBS-T. The membranes were incubated with horseradish peroxidase-conjugated goat
anti-rabbit IgG secondary antibody (1:15,000; Bio-Rad). The protein bands were
visualized using enhanced chemiluminescence (ECL Plus reagent, Amersham
Pharmacia Biotech, Piscataway, NJ) and quantified using densitometry (Sigma Gel,
Jandel Scientific, San Rafael, CA). To control for protein loading, the blots
were stripped using a stripping buffer containing 62.5 mM Tris HCl (pH 6.8),
2% SDS, and 100 mM 2-β-mercaptoethanol, and the blots were
reprobed for β-actin (1:5,000; Abcam, Cambridge, MA) as described
above.
Western blot analysis as previously described (18 (link), 26 (link)). Aliquots of cell
lysate (15-20 μg of protein) were diluted 1:1 with SDS sample buffer,
heated to 95°C for 5 minutes, and electrophoresed on polyacrylamide gel.
Separated proteins were electrotransferred to PVDF membranes, incubated with
5% nonfat milk for one hour, and then incubated overnight with primary
antibody, arginase I (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or
arginase II (1:500; Santa Cruz) overnight and then washed three times with
PBS-T. The membranes were incubated with horseradish peroxidase-conjugated goat
anti-rabbit IgG secondary antibody (1:15,000; Bio-Rad). The protein bands were
visualized using enhanced chemiluminescence (ECL Plus reagent, Amersham
Pharmacia Biotech, Piscataway, NJ) and quantified using densitometry (Sigma Gel,
Jandel Scientific, San Rafael, CA). To control for protein loading, the blots
were stripped using a stripping buffer containing 62.5 mM Tris HCl (pH 6.8),
2% SDS, and 100 mM 2-β-mercaptoethanol, and the blots were
reprobed for β-actin (1:5,000; Abcam, Cambridge, MA) as described
above.
