Fig 7. The pCBS^Ser227 antibody selectively recognizes the CBS phosphorylated form in human urothelium and T24 cells.

(A) Expression of CBS phosphorylated form in human urothelium tissue treated or untreated with 8-Br-cGMP for 15 min. Protein extracts are analyzed by immunoblotting using the anti-pCBS^Ser227 and anti-CBS. Loading in the gel lanes was controlled by detection of GAPDH protein. (B) T24 cells treated or untreated with 8-Br-cGMP for 15 min. Protein extracts are analyzed by immunoblotting using the anti-pCBS^Ser227 and anti-CBS. Loading in the gel lanes was controlled by detection of GAPDH protein. A more robust pCBS specific signal is detected by anti-pCBS^Ser227 in lysates from T24 cells treated with 8-Br-cGMP. No difference in signal intensity is appreciable in the same samples incubated with the anti-CBS. (C) Protein levels are evaluated by densitometric analysis expressed as arbitrary units. (D) Proteins from T24 cells transiently transfected with constructs expressing HA-CBS WT or HA-CBS S227A and treated with 8-Br-cGMP are specifically immunoprecipitated with antibody against the HA epitope. Immunoprecipitates (Ip) are separated by SDS–PAGE and immunoblotted with anti-pCBS^Ser227. Note the absence of signal in cells transfected with HA-CBS S227A mutant construct.