Figure 5. MMC induces phosphorylation of FANCA at S347.
A. Detection of S347-phosphorylated FANCA with a phospho-specific antibody in HA-FANCA overexpressing cells. HEK 293T cells were transfected with pcDNA3-HA-FANCA WT or S347A and then treated with either MMC or the AMPK activator A769662 for 16 h. Cell lysates were immunoprecipitated using an antibody specific to S347-phosphorylated FANCA (P-S347) and immunoblotted with an HRP-conjugated anti-HA antibody (top panel). The presence of equal amounts of HA-FANCA in the inputs was verified by immunoblotting (bottom panel). B. Detection of S347 phosphorylation of endogenous FANCA. HEK 293T cell lysates were treated with 200 ng/mL MMC and then subjected to immunoprecipitation and immunoblotting as described in A, except S347-phosphorylated FANCA was detected using an anti-FANCA antibody (top panel).