(A) Immunoblots showing IP with HA or VHL antibody: A498 cells were co-transfected with ALK5-HA and VHL vectors, followed by TGF-β treatment for 6h, and probed with HA or VHL antibody, respectively (* indicates a spill, Ctrl-1 is isotype specific IgG plus beads, Ctrl -2 is beads only); (B) Immunoblots showing IP with HA or VHL antibody: ACHN cells were transfected with ALK5-HA vector, followed by TGF-β treatment for 6h, and probed with HA or VHL antibody, respectively (Ctrl-1 is isotype specific IgG plus beads, Ctrl -2 is beads only); (C) A498 cells were transiently co-transfected with ALK5-HA and VHL vectors or pcDNA 3. 1(+) empty vector, followed by TGF-β treatment for 6h, and pVHL-ALK5-HA complexes was visualized by staining cells after probing against VHL and HA (red) antibodies followed by PLA. Scale bar 20 μm (mean ± SD of three experiments, 100 cells were analyzed in each group); (D) ACHN cells were transfected with ALK5-HA or pcDNA 3.1(+) empty vector, followed by TGF-β treatment for 6 h, and pVHL-ALK5-HA complexes was visualized by staining cells after probing against VHL and HA (red) antibodies followed by PLA. Scale bar 20 μm (mean ± SD of three experiments, 100 cells were analyzed in each group); (E) Immunoblot (total cell lysates) showing inhibition of proteasomal degradation by proteasomal inhibitor MG132: A498 cells were transfected with ALK5-HA and VHL followed by treatment with TGF-β, or treatment of TGF-β along with MG132 for 6 hours; (F) Immunoblot (total cell lysates) showing inhibition of proteasomal degradation by proteasomal inhibitor MG132: ACHN cells were transfected with ALK5-HA, followed by treatment with TGF-β or treatment with TGF-β along with MG132 for 6 hours; (G) pVHL mediates K48-linked poly-ubiquitination of ALK5: Immunoblot showing enhanced K48-linked poly-ubiquitination of ALK5 by VHL, A498 cells were transfected with indicated plasmids followed by treatment of TGF-β for 6 hours then immunoprecipitated with HA antibody and immunoblot with indicated antibody; (H) PLA analysis and quantification of PLA signals (K48 and HA) in A498 cells transfected with pcDNA 3.1(+) empty vector, ALK5-HA vector alone, or co-transfected with ALK5-HA and VHL vectors (mean ± SD of two experiments, 150 cells were analyzed in each group); (I) pVHL mediates K48-linked poly-ubiquitination of ALK5: Immunoblot showing enhanced K48-linked poly-ubiquitination of ALK5 by VHL, ACHN cells were transfected with indicated plasmids followed by treatment of TGF-β for 6 hours then immunoprecipitated with HA antibody and immune blot with indicated antibody; (J) PLA analysis and quantification of PLA signals (K48- and HA) in ACHN cells transfected with pcDNA 3.1(+) empty vector, ALK5-HA (mean ± SD of two experiments, 150 cells were analyzed in each group).