Fig. 4. Small interfering RNA (siRNA)-mediated knockdown of CBS in HT29 cells. (A) A high expression of the H2S producing enzyme CBS, not cystathionine γ-lyase (CSE), was detected in HT29 cells by immunoblotting analysis. (B) The H2S-producing activities of CBS and CSE were measured by using the methylene blue assay described in the “Experimental” section. 200 μL of the reaction mixture (50 mM HEPES, pH 7.4) contained the cell lysate, 2 mM homocysteine, 2 mM cysteine and either 2 mM dl-propargylglycine (an inhibitor of CSE) or 1 mM aminooxyacetic acid (an inhibitor of CBS and CSE). (C) HT29 cells were transfected with a negative control siRNA (NC siRNA) or a CBS-targeted siRNA (CBS siRNA) for 48 h; then, cell lysates were used for immunoblotting analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control.
