Figure 4
From: OTUD4 enhances TGFβ signalling through regulation of the TGFβ receptor complex
OTUD4 regulates TβRI presence at the plasma membrane. (A) HEK293T cells were transfected with TβRI and either FLAG-OTUD4 or FLAG-OTUD4 DD. Cells were stimulated with TGFβ (100 pM) for the length of time indicated. Cell surface was labelled with biotin at 4 degrees for 40 min before lysis. Lysates were immunoprecipitated with anti- NeutrAvidin affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. β-Actin is used as the loading control. (B) HEK293T cells were transfected with HA-ubiquitin, FLAG-TβRI C.A. and either FLAG-OTUD4 or FLAG-OTUD4 DD. Cells were incubated with MG132 (5 µM) overnight before lysis. After 48 h cells were lysed and immunoprecipitated with anti-TβRI affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. (C) HEK293T cells were transfected with FLAG-TβRI C.A. and either FLAG-OTUD4 or FLAG OTUD4 DD. Whole cell extracts were probed with indicated antibodies. β-Actin is used as the loading control. (D) HEK293T cells were transfected with HA-ubiquitin, FLAG-TβRI C.A. and OTUD4 shRNA constructs B or C. Cells were incubated with MG132 (5 µM) overnight before lysis. After 72 h cells were lysed and immunoprecipitated with anti-TβRI affinity resin. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. (E) HEK293T OTUD4KD1 cells were transfected with FLAG-TβRI C.A. and either HA-ubiquitin (WT), HA-K48 ubiquitin, HA-K63 ubiquitin, HA-K11 ubiquitin, HA-K27 ubiquitin or HA-K29 ubiquitin. Cells were incubated with MG132 (5 µM) overnight before lysis. Immunoprecipitated lysates and whole cell extracts were probed with the indicated antibodies. Full-length blots for all panels are shown in Supplementary Information.
