Fig. 5

ABIN1 negatively regulates the K63-linked polyubiquitination of HIV-1 Tat. a To determine the effect of ABIN1 on the stability of Tat, HEK-293T cells were transfected with Myc-ABIN1 and Flag-Tat encoding plasmids as indicated for 24 h, followed by treatment of CHX at the final concentration of 10 μg/ml for 6 h to arrest cell translation. The levels of Tat protein were detected by Western Blot. b To elucidate whether ABIN1 targeting Tat for proteasome-dependent degradation, HEK-293T cells were transfected with siABIN1-1 or siNC for 24 h, followed by a second round transfection of Flag-Tat encoding plasmids, cells were then treated with MG-132 at the final concentration of 10 ng/ml for 2 h to block proteasomal degradation. The accumulation of Flag-Tat were detected by IB. c The type of ubiquitination regulated by ABIN1 was explored in HEK-293T or HEK-293T^ABIN1-KO cells by immunoprecipitation as in Fig. 4a after transfected with Flag-Tat, together with wild-type HA–Ub, HA–Ub^K63R or HA–Ub^K48R mutant expression vectors for 24 h. β-actin was detected as sample loading control. The blots were representatives of at least three independent experiments achieving similar results