Figure 3.
RIPK1 phosphorylation at S161 occurs prior to RIPK1 phosphorylation at S166. (A)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT, S161E, or S161N mutant and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell survival was measured. Cell lysates were immunoprecipitated (IP) with anti-Flag M2 beads. Total cell lysates (TCL) and IP samples were analyzed by immunoblotting with antibodies as indicated. (B)Ripk1 and Ripk3 double-deficient L929 cells were reconstituted with Flag-RIPK1 WT or S161E mutant and HA-RIPK3 WT and then treated with TNF (10 ng/ml) + zVAD (20 μM) for 2.5 h. Representative confocal and STORM images from two independent experiments of RIPK1 (green) and RIPK3 (purple) in L929 cells with or without treatment are shown. Scale bars, 10 μm for the first and second rows and 200 nm for the third and fourth rows from the top. Area of necrosomes formed after treatment and area ratios of RIPK3 to RIPK1 were analyzed using ImageJ. P values are determined by a two-tailed, unpaired t test. *P < 0.05. (C)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT or S161E mutant and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell lysates were analyzed by immunoblotting to detect proteins as indicated. (D) Cell lysates of nontreated primary MEFs from full-sibling Ripk1+/+ and Ripk1S161E/S161E embryos were analyzed by immunoblotting. Each number represents one embryo. (E) IECs from 8- to 12-wk-old male littermate Ripk1+/+, Ripk1S161E/S161E, and Ripk1S161N/S161N mice injected with PBS or TNF for 6 h were isolated (50 μg/kg of TNF for Ripk1S161E/S161E mice and Ripk1+/+ controls, 400 μg/kg of TNF for Ripk1S161N/S161N mice and Ripk1+/+ controls). Cell lysates were subjected to immunoblotting. (F) Intensities of RIPK1 phosphorylation sites measured by LC–MS/MS. Samples were those in C without or with treatment. (G)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT or mutants (S161E, S161N, S161E + S166A, S161A, and the kinase-dead mutant KK-AT) and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell survival was measured. Cell lysates were analyzed by immunoblotting. Data are presented as mean ± SD of triplicate. The above experiments were independently performed twice. Source data are available for this figure: SourceData F3. Refer to the image caption for details.

RIPK1 phosphorylation at S161 occurs prior to RIPK1 phosphorylation at S166. (A) Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT, S161E, or S161N mutant and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell survival was measured. Cell lysates were immunoprecipitated (IP) with anti-Flag M2 beads. Total cell lysates (TCL) and IP samples were analyzed by immunoblotting with antibodies as indicated. (B)Ripk1 and Ripk3 double-deficient L929 cells were reconstituted with Flag-RIPK1 WT or S161E mutant and HA-RIPK3 WT and then treated with TNF (10 ng/ml) + zVAD (20 μM) for 2.5 h. Representative confocal and STORM images from two independent experiments of RIPK1 (green) and RIPK3 (purple) in L929 cells with or without treatment are shown. Scale bars, 10 μm for the first and second rows and 200 nm for the third and fourth rows from the top. Area of necrosomes formed after treatment and area ratios of RIPK3 to RIPK1 were analyzed using ImageJ. P values are determined by a two-tailed, unpaired t test. *P < 0.05. (C)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT or S161E mutant and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell lysates were analyzed by immunoblotting to detect proteins as indicated. (D) Cell lysates of nontreated primary MEFs from full-sibling Ripk1+/+ and Ripk1S161E/S161E embryos were analyzed by immunoblotting. Each number represents one embryo. (E) IECs from 8- to 12-wk-old male littermate Ripk1+/+, Ripk1S161E/S161E, and Ripk1S161N/S161N mice injected with PBS or TNF for 6 h were isolated (50 μg/kg of TNF for Ripk1S161E/S161E mice and Ripk1+/+ controls, 400 μg/kg of TNF for Ripk1S161N/S161N mice and Ripk1+/+ controls). Cell lysates were subjected to immunoblotting. (F) Intensities of RIPK1 phosphorylation sites measured by LC–MS/MS. Samples were those in C without or with treatment. (G)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT or mutants (S161E, S161N, S161E + S166A, S161A, and the kinase-dead mutant KK-AT) and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell survival was measured. Cell lysates were analyzed by immunoblotting. Data are presented as mean ± SD of triplicate. The above experiments were independently performed twice. Source data are available for this figure: SourceData F3.

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