Figure S3.
RIPK1 phosphorylation at S161 occurs prior to RIPK1 phosphorylation at S166. (A)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 1, 2, or 4 h. Representative 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 third rows and 200 nm for the second 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 < 0.01; *P < 0.05. (B)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT or S161E mutant and then treated with anti-TNF antibody (1 μg/ml) for the indicated time points. Cell lysates were analyzed by immunoblotting to detect proteins as indicated. (C and D) Cell lysates of TNF (10 ng/ml) + zVAD (20 μM)-treated primary BMDMs from littermate Ripk1+/+, Ripk1S161E/S161E, Ripk1S161N/S161N, Ripk1S161A/S161A, and Ripk1K45A/K45A male mice were analyzed by immunoblotting to detect proteins as indicated. (E)Ripk1 KO L929 cells were reconstituted with WT RIPK1 or RIPK1 mutants and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell survival was measured. Cell lysates of nontreated cells were analyzed by immunoblotting to detect proteins as indicated. Data are presented as mean ± SD of triplicate. (F) Intensities of RIPK1 phosphorylation sites measured by LC–MS/MS. Samples were Ripk1 KO L929 cells reconstituted with RIPK1 S166E mutant without treatment. The above experiments were independently performed twice. Source data are available for this figure: SourceData FS3. Refer to the image caption for details.

RIPK1 phosphorylation at S161 occurs prior to RIPK1 phosphorylation at S166. (A) 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 1, 2, or 4 h. Representative 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 third rows and 200 nm for the second 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 < 0.01; *P < 0.05. (B)Ripk1-deficient L929 cells were reconstituted with Flag-RIPK1 WT or S161E mutant and then treated with anti-TNF antibody (1 μg/ml) for the indicated time points. Cell lysates were analyzed by immunoblotting to detect proteins as indicated. (C and D) Cell lysates of TNF (10 ng/ml) + zVAD (20 μM)-treated primary BMDMs from littermate Ripk1+/+, Ripk1S161E/S161E, Ripk1S161N/S161N, Ripk1S161A/S161A, and Ripk1K45A/K45A male mice were analyzed by immunoblotting to detect proteins as indicated. (E)Ripk1 KO L929 cells were reconstituted with WT RIPK1 or RIPK1 mutants and then treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Cell survival was measured. Cell lysates of nontreated cells were analyzed by immunoblotting to detect proteins as indicated. Data are presented as mean ± SD of triplicate. (F) Intensities of RIPK1 phosphorylation sites measured by LC–MS/MS. Samples were Ripk1 KO L929 cells reconstituted with RIPK1 S166E mutant without treatment. The above experiments were independently performed twice. Source data are available for this figure: SourceData FS3.

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