Fig. 3
From: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP
ERAP1 promotes βTrCP ubiquitylation by interacting with USP47. a–d MEFs were transfected with ERAP1 and/or Flag-USP47. Interaction between USP47 and ERAP1 was detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. e MEFs transfected with ERAP1 were stained with anti-ERAP1 and anti-USP47 antibodies. Green and red, USP47 and ERAP1 expressing cells, respectively. Nuclei were counter stained with Hoechst (Blue). Magnification ×60; Bars: 5 μm. Representative images from three independent experiments. f βTrCP and Gli steady state in USP47+/+ and USP47−/− MEFs. g βTrCP protein level in USP47+/+, USP47−/− and USP47−/− Flag-USP47 transfected MEFs. h βTrCP half-life in USP47+/+ vs. USP47−/− MEFs treated with CHX (100 µg/mL) at the indicated times. i βTrCP protein levels in MEFs transfected with empty vector as control or Flag-USP47 and treated with CHX (100 µg/mL) at different time points. j Gli1 protein levels in Ptch−/− MEFs transfected with empty vector as control or Flag-USP47 and treated after 24 h with CHX (100 µg/mL) for different time points. In g–j densitometric analysis of βTrCP and Gli1 protein levels of three independent experiments are shown (right panels). k MEFs were transfected with HA-Ub and increasing amount of ERAP1 in the presence or absence of Flag-USP47. Endogenous βTrCP was immunoprecipitated with an anti-βTrCP antibody and the ubiquitylated forms were revealed with an anti-HA antibody (upper panel). The blot was reprobed with an anti-βTrCP antibody. Flag-USP47, ERAP1 and βTrCP total protein levels are shown (lower panel). l MEFs were transfected with Flag-USP47 and increasing amount of ERAP1. Interaction between Flag-USP47 and endogenous βTrCP was assessed by immunoprecipitation and immunoblotting with the indicated antibodies. Actin was used as loading control. m MEFs were transfected with Flag-USP47 and treated for 24 h with Leu-SH at the indicated concentration. Interaction between Flag-USP47 and endogenous βTrCP was detected as described in l. Densitometric analysis of the Flag-USP47/βTrCP binding ratio representative of three independent experiments is shown (right panel). n MEFs were transduced with shCTRL or shERAP1 and transfected with Flag-USP47. Interaction between Flag-USP47 and endogenous βTrCP was assessed as described in l
