FIG 6.

Inactivation of PP5 correlates with the suppression of DNA-PK activity. (A) 293SLAM cells were infected with MeV for 24 h and then immunoprecipitated with anti-DNA-PKcs, anti-PP2A, anti-PP5, or anti-PP6c antibody. Phosphatase activity in immunocomplexes was determined by an in vitro phosphatase assay. The results are expressed as values relative to the value of the mock-treated control sample (upper panel). The protein level of each protein was detected by immunoblotting (lower panel). (B) Knockdown of PP5 by RNA interference. 293SLAM cells were transfected with siRNA targeting PP5. For controls, cells were either mock treated or transfected with a nonspecific mismatched control siRNA (ctrl). After 48 h, the cells were lysed and subjected to immunoblotting or EMSA, or they were radiolabeled with ³²PO₄ and immunoprecipitated with anti-Sp1 antibody. (C) The cell lysates in panel B were subjected to immunoblotting with anti-c-Myc and anti-PP5 antibodies. (D) Total RNA prepared from cells transfected with ctrl siRNA or siRNA targeting PP5 described in panel B were then subjected to qPCR. The expression level in PP5 knock down cells was represented as a value relative to the that of ctrl siRNA-transfected cells. The specific primer pairs used are indicated by numbered columns as follows: 1, RNA18S5; 2, HIST1H2AC; 3, CASP3; 4, JAK1; 5, MAPK6; 6, IRF1; 7, NDUFB4; 8, COX7C; 9, RPL23; 10, LAMA4; 11, HSPA1L; and 12, ATP5G3. The official full names of these genes are described in the supplemental material.