PDLIM2 promotes K48-linked ubiquitination and proteasomal degradation of Tax. (A) 293 cells were transfected with Tax in the presence or absence of Myc-PDLIM2, followed by IP using Myc antibody and IB using Tax antibody. The expression levels of Tax and Myc-PDLIM2 were analyzed by direct IB. (B) 293 cells transfected with Myc-PDLIM2 alone or together with Tax were subjected to IP using Tax antibody and IB using Myc antibody. The expression levels of Tax and Myc-PDLIM2 were also analyzed. (C) Jurkat and C8166 cells were subjected to IP using PDLIM2 antibody (lanes 1 and 3) or control IgG (lanes 2 and 4) and IB using Tax antibody. The input was monitored by direct IB using Tax, PDLIM2, or Hsp90 antibody. (D) 293 cells were transfected with Tax and GFP in the presence of increasing amounts of PDLIM2, followed by IB to detect expression levels of Tax, GFP, PDLIM2, and Hsp90. (E) 293 cells transfected with Tax in the presence or absence of PDLIM2 were CHX chased at the indicated time (in hours). In lanes 4 and 8, the cells were chased in the presence of 10 μM MG132. The protein levels of Tax, Myc-PDLIM2, and Hsp90 were analyzed by direct IB. (F) MT-4 cells stably expressing PDLIM2 or an empty vector were CHX chased as described in panel D. (G) 293 cells transfected with the indicated constructs were left untreated or treated with MG132 for 3 hours, followed by IP-IB to detect the ubiquitinated Tax in the nucleus (top). To better compare Tax ubiquitination, the similar amounts of Tax proteins were used in each lane for IP (middle). The expression levels of PDLIM2 and its LIM deletion mutants were analyzed by IB (bottom).