Fig 6. Mutation of Ser 227 impairs PKG-mediated increase of H₂S production on T24 cells.

(A) Western blot analysis of proteins from CBSΔT24 cells transiently transfected with plasmids expressing HA-CBS WT and HA-CBS mutants (HA-CBS S227A, HA-CBS S525A), incubated with 8-Br-cGMP or vehicle for 15 min. Loading in the gel lanes was controlled by detection of GAPDH protein. (B) Production of H₂S from the same samples. Mutation of Ser227 significantly reduces H₂S production in basal condition and following incubation with 8-Br-cGMP compared with the paired WT (^#p<0.05 and ^###p<0.001). The levels of H₂S are not affected by the mutation of Ser 525. Indeed, H₂S levels are similar to those obtained from HA-CBS WT either in basal condition or upon cyclic nucleotide stimulation. 8-Br-cGMP significantly increases H₂S production in HA-CBS WT and in HA-CBS S525A compared to vehicle (*p<0.05, ^§§§p<0.001). Data are calculated as nanomoles per milligram of protein per min and expressed as mean±SE of three different experiments.