Fig. 7.
TRIM44 deubiquitinates HIF-1α via zinc finger domains. a TRIM44OE RPMI, U266, and 293T cells were cultured in hypoxia for 4 h. The cells were immunoprecipitated using IgG or HIF-1α antibodies and were then immunoblotted using TRIM44 or HIF-1α antibodies. b Diagram of functional domains contained in truncated TRIM44. c Identification of the TRIM44 functional domain for HIF-1α deubiquitination. A combination of HA-HIF-1α, His-Ub, and various truncated forms of TRIM44-GFP (Full, ZF:zinc finger only, BB: B box domain only, CC: coiled coil domain only, ZF-BB: zinc finger and BB domains, BB-CC: BB domain and CC domain) were expressed in 293T cells. The cells were treated with MG132 for 6 h before collection. Ubiquitinated HIF-1α (over 120 kD) was measured by immunoblotting. d 293T cells were transfected with HA-HIF-1α combined with full length TRIM44 (FL), or the ZF, BB, or CC domain of TRIM44. After 48 h, the cells were treated for 0, 30, or 60 min with cycloheximide (CHX). HIF-1α levels were determined by immunoblotting and were normalized by β-actin. e 293T cells were transfected with HA-HIF-1α in the presence of the ZF-UBP domain (ZF), B-box domain (BB), or coiled-coil domain (CC) of TRIM44. Cell lysates were immunoprecipitated with anti-GFP. Immunoblotting was performed to determine the interaction between HIF-1α and the TRIM44 domains