Journal: Aging Cell

Article Title: CDK2 phosphorylation of Werner protein (WRN) contributes to WRN’s DNA double‐strand break repair pathway choice

doi: 10.1111/acel.13484

Figure Lengend Snippet: CDK2‐CycE phosphorylates WRN. (a) FLAG/His‐tagged WRN protein was purified from baculovirus‐infected insect cells. The purified WRN protein was in vitro phosphorylated with the indicated CDK‐Cyc combinations and probed by Western blot for the antigens shown. Phosphorylated serine/threonine blot intensities were quantified by normalizing with WRN and basal phosphorylation without CDKs. Asterisks indicated directly above the columns represent p‐value calculations compared to controls (basal phosphorylation without CDKs). (b) To remove the basal phosphorylation seen in A, purified WRN proteins were treated with λPPase, then immunoprecipitated with FLAG beads and subjected to in vitro phosphorylation with CDK2‐CycA or CDK2‐Cyc2 combinations, followed by Western blot analysis. Phosphorylated serine blot intensity was quantified by normalizing with WRN. Error bars represent standard deviation. p ‐value, *, <0.05; **, <0.01; ***, <0.001

Article Snippet: Proteins were resolved in 4 to 15% Mini‐PROTEAN TGX gels (BioRad, Hercules, CA, USA), electroblotted to nitrocellulose membrane, and visualized using antibodies against anti‐WRN (in house), anti‐phospho‐CDK substrate (Cell Signaling, 9477), anti‐CDK1 (Abcam, ab133327), anti‐CDK2 (Abcam, ab32147), anti‐CycA2 (Abcam, ab38), anti‐CycE (Abcam, ab133266), anti‐DNA‐PKcs (BD, 610804), anti‐KU70 (ThermoFisher, PA5‐27538), and anti‐RPA (Calbiochem, NA18).

Techniques: Purification, Infection, In Vitro, Western Blot, Immunoprecipitation, Standard Deviation