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. 2020 Sep 16;10(25):11520–11534. doi: 10.7150/thno.44829

Figure 3.

Figure 3

CLU competes against TAK1 for binding TGFBR1. (A) and (B) Reciprocal immunoprecipitation showing interaction between TGFBR1 and CLU. 293T cells were co-transfected with CLU-3×Flag and TGFBR1-myc expressing plasmid, followed by Co-IP with Flag (A) or myc (B) antibody. (C) 293T-tet-CLU cells were co-transferred with TRAF6-HA and TGFBR1-Myc expressing plasmids as indicated. Cell lysates were IPed with Myc antibody followed by western blotting with HA, Myc and CLU antibodies respectively. (D) H460-tet-CLU cells were co-transferred with TRAF6-HA and TGFBR1-Myc expressing plasmids as indicated. Cell lysates were IPed with Myc antibody followed by western blotting with HA, Myc and CLU antibodies respectively. (E) 293T-tet-CLU cell were co-transfected with TAK1-Flag and TGFBR1-Myc expressing plasmids. Cell lysates IPed with Flag antibody followed by western blotting with Myc, Flag and CLU antibodies respectively. (F) H460-tet-CLU cell lysate IPed with TAK1 antibody followed by western blotting with TGFBR1, TAK1 and CLU antibodies. (G) 293T-tet-CLU cells were co-transfected with TAK-Flag and TAB2-Myc expressing plasmids as indicated. Cell lysates were IPed with Myc antibody followed by western blotting with Flag, Myc and CLU antibodies respectively. (H) Schematic diagram of TGFBR1-myc truncation mutation. (I) Co-IP evaluating the interaction between CLU and TGFBR1 or its mutants. 293T cells were co-transfected with CLU-3×Flag and TGFBR1-myc or its muctant expressing plasmid for 24 h before performing Co-IP with Myc antibody and immunoblotting with Flag and Myc tag antibodies.