FIG 6.

RTA reduces the protein level of GBP1 through proteasome-mediated degradation. (A) HEK 293T cells were transfected with HA-GBP1 and increasing amounts of RTA-SF. After 36 h, cell lysates were subjected to immunoblotting. (B) iSLK-GBP1 cells were established as described in Materials and Methods and were treated with or without 5 μg/ml DOX for the induction of RTA. Cell lysates were harvested at the indicated time points and analyzed by Western blotting. (C) HeLa cells were transfected with RTA-SF or the vector control. After 36 h, RNA was isolated for qPCR. (D) HEK 293T cells were transfected with HA-GBP1 with or without RTA-SF and treated with 10 μM MG132 as indicated. Anti-HA and anti-Flag antibodies were used for Western blotting. (E) HEK 293T cells were cotransfected with HA-GBP1 and RTA mutants. After 36 h, cells were collected for subsequent Western blotting. (F) RTA-SF and HA-GBP1 were transfected into HEK 293T cells as indicated. After 48 h, cells were lysed and immunoprecipitated by using anti-Flag M2 affinity gel. The purified proteins, along with input samples, were detected by Western blotting.