Figure 2.

ATR-dependent WRN phosphorylation upon Aph treatment. (A) WB detection of WRN phosphorylation in HEK293T cells treated or not with 0.4 μM Aph for the indicated time-points. Cell extracts were immunoprecipitated using anti-WRN antibody followed by immunoblotting with an anti-S/TQ antibody. Total WRN was used to assess the amount of WRN immunoprecipitated. The ratio of phosphorylated protein to total protein is reported below each lane. (B) Analysis of WRN status in HEK293T cells transfected with plasmids expressing the Flag-tagged full-length wild-type WRN (wt) or a full-length carrying Ala substitutions at all the six S/TQ sites (6A) and treated or not with Aph as indicated. Cell extracts were prepared 48 h post-transfection and used to immunoprecipitate ectopic WRN with anti-Flag tag antibody, followed by immunoblotting with an anti-S/TQ antibody. Total WRN was used to assess the amount of wild-type or mutant form of WRN immunoprecipitated. All experiments are representative images of at least two replicates.