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. 2012 May 18;33(6):563–574. doi: 10.1007/s10059-012-2294-1

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Fig. 5. — PKCδ mediates the effect of HA on activation of TGFβ receptor signaling, and the induction of PAI-1 by HA occurs independently of Smad2. (A) HUVECs were treated with or without HA (1 mDa, 200 μg/ml) for various time intervals. Cell lysates were immunoprecipitated with anti-TGFβRI antibody (2 μg/ml), followed by Western blot analysis (upper panel). HUVECs were treated with HA (1 mDa, 200 μg/ml) or human recombinant TGFβ1 (Sigma, 50 ng/ml) for 1 h. To determine effect of TGFβRII on activation of TGFβRI, HUVECs were pre-incubated with soluble form of TGFβRII (TGFβRII-Fc chimera, 50 ng/ml) for 3 h, prior to incubation with HA (1 mDa, 200 μg/ml) or TGFβ1 (50 ng/ml). Cell lysates were prepared and immunoprecipitated with anti-actin antibody (2 μg/ml) or anti-TGFβRI antibody (2 μg/ml), followed by Western blot analysis (lower panel). Cell lysates prepared were also subjected to western blot analysis (lower panel). The rTGFβ denotes human recombinant TGFβ and sTGFβRII denotes the soluble form of TGFβRII. (B) HUVECs were pretreated with or without various concentrations of SB431542, an inhibitor of TGFβRI, for 30 min, followed by treatment with or without HA (1 mDa, 200 μg/ml) for 1 h. Cell lysates were then subjected to Western blot analysis (upper panel). HUVECs were transfected with control SiRNA (10 nM) or RHAMM SiRNA (10 nM). At 48 h after transfection, cells were pretreated with or without SB431542 (1 μM) for 30 min, followed by treatment with or without HA (1 mDa, 200 μg/ml) for 1 h. Cell lysates were prepared and immunoprecipitated with anti-TGFβRI antibody, followed by Western blot analysis (lower panel). C denotes control SiRNA and R denotes RHAMM SiRNA. (C) HUVECs were transiently transfecetd with control vector (1 μg) or dominant negative PKCδ (1 μg). At 48 h after transfection, cell lysates were immunoprecipitated with anti-actin antibody (2 μg/ml) or anti-TGFβRI antibody (2 μg/ml), followed by Western blot analysis using the indicated antibodies (upper panel). Cell lysates were also subjected to Western blot analysis (lower panel).