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. Author manuscript; available in PMC: 2010 Jun 7.

Published in final edited form as: J Cell Physiol. 2009 Dec;221(3):707–715. doi: 10.1002/jcp.21910

Fig. 1 — A: 293T cells were transiently transfected with km23-2-Flag, RI-V5, RII-HA, and/or EV, followed by IP/blot analyses using an anti-Flag Ab as the IP Ab and an RII polyclonal Ab as the blotting Ab (top panel). Cells were incubated in SF medium for 1h prior to addition of TGFb (10 ng/ml) for 5 min. Lower panels, controls for expression and loading of RI (V5 blot), RII (RII blot), and km23-2 (Flag blot). The results shown are representative of two similar experiments. B: HaCaT cells were transiently transfected either EV (lane 1) or km23-2-Flag (lanes 2-7). 24h after transfection, HaCaT cells were then incubated with SF medium for I h prior to addition of TGFβ (5 ng/ml) for the indicated times. Top panel, cells were then lysed , and lysates were subjected to IP/blot analyses using a Flag Ab as the IP Ab and an RII Ab as the blotting Ab. Bottom panels, Western blot analysis to demonstrate equal loading and expression of endogenous RII and km23-2-Flag. C: 293T cells were transiently transfected with RI-V5, RII-HA or KNRII, and either EV or km23-2-Flag. 28 h after transfection, in vivo phosphorylation of km23-2 were performed as described previously (Tang et al., 2002). Lower panel, the gel from top panel was transferred to a PVDF membrane and blotted with anti-Flag Ab. D: Mv1Lu, R1B (Mv1Lu cells lacking functional TβRI), and DR26 (Mv1Lu cells containing a truncated, nonfunctional TβRII) (Laiho et al., 1991; Laiho et al., 1990b) were transiently transfected with km23-2-Flag. 28h after transfection, in vivo phosphorylation assays were performed as described previously (Tang et al., 2002). Lower panel, the gel from top panel was transferred to a PVDF membrane and blotted with an anti-Flag Ab. Results are representative of two experiments.

Fig. 1 — A: 293T cells were transiently transfected with km23-2-Flag, RI-V5, RII-HA, and/or EV, followed by IP/blot analyses using an anti-Flag Ab as the IP Ab and an RII polyclonal Ab as the blotting Ab (top panel). Cells were incubated in SF medium for 1h prior to addition of TGFb (10 ng/ml) for 5 min. Lower panels, controls for expression and loading of RI (V5 blot), RII (RII blot), and km23-2 (Flag blot). The results shown are representative of two similar experiments. B: HaCaT cells were transiently transfected either EV (lane 1) or km23-2-Flag (lanes 2-7). 24h after transfection, HaCaT cells were then incubated with SF medium for I h prior to addition of TGFβ (5 ng/ml) for the indicated times. Top panel, cells were then lysed , and lysates were subjected to IP/blot analyses using a Flag Ab as the IP Ab and an RII Ab as the blotting Ab. Bottom panels, Western blot analysis to demonstrate equal loading and expression of endogenous RII and km23-2-Flag. C: 293T cells were transiently transfected with RI-V5, RII-HA or KNRII, and either EV or km23-2-Flag. 28 h after transfection, in vivo phosphorylation of km23-2 were performed as described previously (Tang et al., 2002). Lower panel, the gel from top panel was transferred to a PVDF membrane and blotted with anti-Flag Ab. D: Mv1Lu, R1B (Mv1Lu cells lacking functional TβRI), and DR26 (Mv1Lu cells containing a truncated, nonfunctional TβRII) (Laiho et al., 1991; Laiho et al., 1990b) were transiently transfected with km23-2-Flag. 28h after transfection, in vivo phosphorylation assays were performed as described previously (Tang et al., 2002). Lower panel, the gel from top panel was transferred to a PVDF membrane and blotted with an anti-Flag Ab. Results are representative of two experiments.