Fig. 7

MAP2K7 degrades the SVCV P protein by attenuating USP7-mediated K63-linked polyubiquitination. (A) EPC cells were cotransfected with the indicated plasmids (1 ?g each). At 18 h after transfection, the cells were treated with dimethyl sulfoxide (DMSO), MG132 (20 mM), 3-MA (2 mM), NH4Cl (20 mM), or chloroquine (CQ) (100 mM) for 6 h before IB analysis was performed. (B) EPC cells were cotransfected with the indicated plasmids (1 ?g each). At 18 h after transfection, the cells were treated with DMSO or MG132 (5, 10, or 20 mM) for 6 h. (C and D) MAP2K7 decreases the ubiquitination of the SVCV P protein. EPC cells were transfected with 5 ?g P-Flag, 5 ?g MAP2K7-HA or empty vector, and 2 ?g Ub-related plasmids. At 18 hpt, the cells were treated with DMSO or MG132 for 6 h. Cell lysates were IP with anti-Flag affinity gels. (E and F). EPC cells transfected with the indicated plasmids (5 ?g each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-HA affinity gels. (G) EPC cells were cotransfected with the 1 ?g P-Flag and USP7-Myc (0,0.5 or 1 ?g). (H) EPC cells were cotransfected with the 1 ?g P-Flag, 1 ?g MAP2K7-HA, and USP7-Myc (0, 0.25, 0.5 or 1 ?g). (I) EPC cells were cotransfected with the 1 ?g USP7-Myc and 1 ?g sh-USP7#1 (or sh-USP7#2). (J) EPC cells were cotransfected with the 1 ?g P-Flag, 1 ?g MAP2K7-HA, and sh-USP7#1 (0, 0.25, 0.5, or 1 ?g).