Fig. 3.
Rep52 and Rep78 mediate phosphorylation of KAP1-S824 through interactions with PP1. (A) p-KAP1-S824 in 293T cells infected with Ad5 alone or coinfected with Ad5 and either AAV2 or rAAV2 [1,000 genome-containing particles (gcp) per cell] monitored at 4, 18, 24, and 42 h post infection. (B) p-KAP1-S824 in 293T cells expressing the indicated Rep proteins. Values are reported as mean ± SEM, n = 3. (C) GFP-trap experiment performed in 293T cells expressing Rep52 and GFP-tagged PP1α, PP1β, PP1γ, or a GFP control. (D–F) AAV2 replication and transcription at different time points in 293T cells depleted for PP1α (siPP1α), PP1β (siPP1β), or both (siPP1α/β). (D) PP1 depletion analyzed by Western blotting. (E) AAV2 genome replication. Values are reported as mean ± SEM, n = 7. (F) Transcription from the three AAV2 promoters. Values are reported as mean ± SEM, n = 4. (G) Coimmunoprecipitation of FLAG-tagged proteins from 293T cells expressing FLAG-PP1α or a FLAG-GFP control and the indicated Rep proteins. (H) Immunoblot of p-KAP1-S824 in 293T cells transfected with either Rep52 or Rep52K372A. Protein levels were quantified using ImageJ software. Values are reported as mean ± SEM, n = 3. Statistical significance was determined by unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001. EV, empty vector; IP, immunoprecipitation.