Figure 5.
Gab2/PLCγ2/PKC axis mediates IL-1β–induced activation of CARMA3. (A) IL-1β treatment induces the phosphorylation of CARMA3 in endothelial cells. HUVEC were treated with a control vehicle or IL-1β for 5 minutes. The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, and the CARMA3 was immunoprecipitated using polyclonal CARMA3 antibodies. The immunoprecipitates were subjected to immunoblot analysis and probed with monoclonal antibodies against phosphoserine. (B) Gab2 or PLCγ2 silencing blocks IL-1β–induced phosphorylation of CARMA3. HUVECs transfected with scRNA, Gab2 siRNA, or PLCγ2 siRNA were treated with a control vehicle or IL-1β for 5 minutes. The cell lysates were immunoprecipitated with CARMA3 antibodies, and the immunoprecipitates were probed for the presence of phosphorylated CARMA3, as described in the legend to panel (A). (C) IL-1β induces the activation of PLCγ2 and PKC isomers in endothelial cells. HUVECs were treated with a control vehicle or IL-1β for 5 minutes. At the end of the 5-minute treatment, the cell lysates were harvested and probed for the phosphorylation of PLCγ2, PKCα/β, and PKCδ by immunoblot analysis using antibodies against phosphorylated PLCγ2, PKCα/β, and PKCδ. (D) Gab2 silencing inhibits IL-1β–induced activation of PLCγ2 and PKC isomers in endothelial cells. HUVECs were transfected with scRNA or Gab2 siRNA (200 nM). After 48 hours, the Gab2 knockdown was confirmed by immunoblot analysis. HUVECs transfected with scRNA or Gab2 siRNA were treated with a control vehicle or IL-1β for 5 minutes. The cell lysates were probed for the presence of phosphorylated PLCγ2, PKCα/β, and PKCδ by immunoblot analysis. (E) PLCγ2 silencing blocks the IL-1β–induced activation of PKC isomers. HUVECs were transfected with scRNA or PLCγ2 siRNA. After 48 hours, PLCγ2 knockdown was confirmed by immunoblotting. HUVECs transfected with scRNA or PLCγ2 siRNA were treated with a control vehicle or IL-1β for 5 minutes. The phosphorylation of PKCα/β and PKCδ were analyzed by immunoblot analysis. (F) Inhibition of PKC attenuates IL-1β–induced phosphorylation of CARMA3. HUVECs were treated with PKC α/β inhibitor Go6976 (Go; 100 nM), pan-PKC inhibitor bisindolylmaleimide 1 (BisM-1; 500 nM), or DMSO vehicle for 1 hour. After that, the cells were treated with a control vehicle or IL-1β for 5 minutes. The cell lysates were processed as described in the panel (D) and probed for the phosphorylation of CARMA3. The immunoblots shown were the representative blots of 3 independent experiments with similar results.

Gab2/PLCγ2/PKC axis mediates IL-1β–induced activation of CARMA3. (A) IL-1β treatment induces the phosphorylation of CARMA3 in endothelial cells. HUVEC were treated with a control vehicle or IL-1β for 5 minutes. The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, and the CARMA3 was immunoprecipitated using polyclonal CARMA3 antibodies. The immunoprecipitates were subjected to immunoblot analysis and probed with monoclonal antibodies against phosphoserine. (B) Gab2 or PLCγ2 silencing blocks IL-1β–induced phosphorylation of CARMA3. HUVECs transfected with scRNA, Gab2 siRNA, or PLCγ2 siRNA were treated with a control vehicle or IL-1β for 5 minutes. The cell lysates were immunoprecipitated with CARMA3 antibodies, and the immunoprecipitates were probed for the presence of phosphorylated CARMA3, as described in the legend to panel (A). (C) IL-1β induces the activation of PLCγ2 and PKC isomers in endothelial cells. HUVECs were treated with a control vehicle or IL-1β for 5 minutes. At the end of the 5-minute treatment, the cell lysates were harvested and probed for the phosphorylation of PLCγ2, PKCα/β, and PKCδ by immunoblot analysis using antibodies against phosphorylated PLCγ2, PKCα/β, and PKCδ. (D) Gab2 silencing inhibits IL-1β–induced activation of PLCγ2 and PKC isomers in endothelial cells. HUVECs were transfected with scRNA or Gab2 siRNA (200 nM). After 48 hours, the Gab2 knockdown was confirmed by immunoblot analysis. HUVECs transfected with scRNA or Gab2 siRNA were treated with a control vehicle or IL-1β for 5 minutes. The cell lysates were probed for the presence of phosphorylated PLCγ2, PKCα/β, and PKCδ by immunoblot analysis. (E) PLCγ2 silencing blocks the IL-1β–induced activation of PKC isomers. HUVECs were transfected with scRNA or PLCγ2 siRNA. After 48 hours, PLCγ2 knockdown was confirmed by immunoblotting. HUVECs transfected with scRNA or PLCγ2 siRNA were treated with a control vehicle or IL-1β for 5 minutes. The phosphorylation of PKCα/β and PKCδ were analyzed by immunoblot analysis. (F) Inhibition of PKC attenuates IL-1β–induced phosphorylation of CARMA3. HUVECs were treated with PKC α/β inhibitor Go6976 (Go; 100 nM), pan-PKC inhibitor bisindolylmaleimide 1 (BisM-1; 500 nM), or DMSO vehicle for 1 hour. After that, the cells were treated with a control vehicle or IL-1β for 5 minutes. The cell lysates were processed as described in the panel (D) and probed for the phosphorylation of CARMA3. The immunoblots shown were the representative blots of 3 independent experiments with similar results.

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