Fig. 2.

CO activates protein methylation via CBS activity. A: U937 cell extracts were prepared from grown in the presence of 0, 25, 50 or 100 µM CO-releasing molecule (CORM) 2 for 8 h. Increased methylation level of protein was evaluated with ASYM24, anti-asymmetric dimethylarginine antibody. Asterisks indicate the bands changing the intensity with CORM-treatment. B: U937 cells were treated with 0, 5, 10, 20 µM hemin for 8 h. Cell lysate (25 µg) was separated by SDS-PAGE and immunoblotted with ASYM24 antibody and anti-heme oxygenase-1 (HO-1) antibody. Asterisks indicate the bands changing the intensity with hemin-treatment. C: U937 cells were exposed with 100 µM CORM for 0, 0.5, 1, 2, 4, 6 or 8 h. Extracted protein was separated by SDS-PAGE, and immunoblotted with ASYM24 antibody. Asterisks indicate the bands changing the intensity with CORM-treatment. D: U937 cells were transfected with 3 µg of siRNA for non-targeting or CBS for 48 h as described in Materials and Methods. After further treatment with 100 mM of Ruthenium chloride (III) (Ru) or CORM-2 (CO) for 6 h, levels of total protein methylation were detected using western blotting with ASYM24 antibody. The expression level of GAPDH was determined as an internal control. Asterisks indicate the bands changing the intensity with CORM-treatment. These are representative pictures of 5 separate experiments.