Figure 4
The FasL ICD dampens ERK1/2 activity during B cell activation by inhibiting PKCα phosphorylation. (A) Increased ERK1/2 phosphorylation in activated homozygous FasL ΔIntra B cells. Naive splenic B cells were stimulated with 1 μg/mL soluble anti-IgM antibody for the indicated time points and analyzed for ERK1/2 phosphorylation by ELISA. The diagram displays the relative induction of ERK1/2 activation over time during anti-IgM treatment, measured as the increase in relative fluorescence intensity of phosphorylated ERK1/2 compared with total ERK1/2 protein. Wild-type cells are represented by the dark gray columns and homozygous FasL ΔIntra cells by light gray columns. Mean values of 4 independent experiments are shown, and the error bars indicate the SE. (B) Western Blot analysis of total cell lysates prepared from wild-type and homozygous FasL ΔIntra splenic B cells that were stimulated for 0, 5, 10, and 30 minutes with 1 μg/mL anti-IgM antibody. Phosphorylated ERK1/2 was detected with a rabbit anti–mouse pERK1/2 antibody; β-actin served as a loading control. (C,D) The activating phosphorylation of PLCγ2 and PKCα is repressed by the FasL ICD. Activation of PLCγ2 (C) and PKCα (D) after stimulation of naive wild-type and homozygous FasL ΔIntra splenic B cells with 25 μg/mL F(ab′)2 anti-IgM antibody for the indicated times was evaluated by intracellular staining of the phosphorylated proteins, followed by FACS analysis. The relative geometric mean fluorescent intensity (MFI) of activated cells was calculated against the MFI of untreated cells. Mean data of 11 (PLCγ2) and 12 (PKCα) independent experiments are shown together with standard errors. (E) ERK1/2 activation in activated mouse B cells is PKCα-dependent. ERK phosphorylation, in response to B cell stimulation with 1 μg/mL anti-IgM antibody, was determined by ELISA, either in the presence or absence of the PKC inhibitor GF109203x hydrochloride (1μM). Wild-type cells are illustrated by solid and homozygous FasL ΔIntra cells by striped columns. The gray color represents cells without and the black color the corresponding inhibitor-treated cells. (F) PKCα regulates FasL reverse signaling-influenced B cell proliferation. Proliferation of wild-type and homozygous FasL ΔIntra naive splenic B cells in response to stimulation with 100 ng/mL PMA and 0.2 μg/mL ionomycine in the presence (1μM) or absence of the PKC inhibitor GF109203x hydrochloride (Sigma-Aldrich) was measured after 72 hours by a FACS-based CFSE dilution assay. Nonstimulated cells are represented by the light gray-filled histogram. The solid line shows activated B cells without PKC inhibitor, the dotted line indicates the activated inhibitor-treated cells. One representative experiment of 4 is shown.

The FasL ICD dampens ERK1/2 activity during B cell activation by inhibiting PKCα phosphorylation. (A) Increased ERK1/2 phosphorylation in activated homozygous FasL ΔIntra B cells. Naive splenic B cells were stimulated with 1 μg/mL soluble anti-IgM antibody for the indicated time points and analyzed for ERK1/2 phosphorylation by ELISA. The diagram displays the relative induction of ERK1/2 activation over time during anti-IgM treatment, measured as the increase in relative fluorescence intensity of phosphorylated ERK1/2 compared with total ERK1/2 protein. Wild-type cells are represented by the dark gray columns and homozygous FasL ΔIntra cells by light gray columns. Mean values of 4 independent experiments are shown, and the error bars indicate the SE. (B) Western Blot analysis of total cell lysates prepared from wild-type and homozygous FasL ΔIntra splenic B cells that were stimulated for 0, 5, 10, and 30 minutes with 1 μg/mL anti-IgM antibody. Phosphorylated ERK1/2 was detected with a rabbit anti–mouse pERK1/2 antibody; β-actin served as a loading control. (C,D) The activating phosphorylation of PLCγ2 and PKCα is repressed by the FasL ICD. Activation of PLCγ2 (C) and PKCα (D) after stimulation of naive wild-type and homozygous FasL ΔIntra splenic B cells with 25 μg/mL F(ab′)2 anti-IgM antibody for the indicated times was evaluated by intracellular staining of the phosphorylated proteins, followed by FACS analysis. The relative geometric mean fluorescent intensity (MFI) of activated cells was calculated against the MFI of untreated cells. Mean data of 11 (PLCγ2) and 12 (PKCα) independent experiments are shown together with standard errors. (E) ERK1/2 activation in activated mouse B cells is PKCα-dependent. ERK phosphorylation, in response to B cell stimulation with 1 μg/mL anti-IgM antibody, was determined by ELISA, either in the presence or absence of the PKC inhibitor GF109203x hydrochloride (1μM). Wild-type cells are illustrated by solid and homozygous FasL ΔIntra cells by striped columns. The gray color represents cells without and the black color the corresponding inhibitor-treated cells. (F) PKCα regulates FasL reverse signaling-influenced B cell proliferation. Proliferation of wild-type and homozygous FasL ΔIntra naive splenic B cells in response to stimulation with 100 ng/mL PMA and 0.2 μg/mL ionomycine in the presence (1μM) or absence of the PKC inhibitor GF109203x hydrochloride (Sigma-Aldrich) was measured after 72 hours by a FACS-based CFSE dilution assay. Nonstimulated cells are represented by the light gray-filled histogram. The solid line shows activated B cells without PKC inhibitor, the dotted line indicates the activated inhibitor-treated cells. One representative experiment of 4 is shown.

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