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. 2022 Jun 3;32(7):621–637. doi: 10.1038/s41422-022-00673-3

Fig. 1. Activated RIPK1 can be found in nucleus.

Fig. 1

a, b Wild-type MEFs were treated as indicated and the fraction of nucleus or cytoplasm was analyzed by western blotting. 5 ng/mL mTNFα, 50 µM SM164, 100 nM (5Z)-7-oxozeaeno, 20 µM zVAD-fmk were used. mTNFα was added 0.5 h after the addition of other compounds and treated for the indicated period. c, e, f, g Wild-type, Ripk1D138N/D138N, Ripk3−/−, Mlkl−/−, or Ripk1D325A/D325A MEFs were pre-incubated with 50 µM SM164, 20 µM zVAD-fmk, with or without 10 µM Nec-1s for 0.5 h and then treated with 5 ng/mL mTNFα. The levels of p-S166-RIPK1, RIPK1, RIPK3, MLKL in the fractions of nucleus and cytoplasm were determined by western blotting. d Immunostaining of p-S166 RIPK1 (red) in wild-type and Ripk1D138N/D138N MEFs treated with 50 µM SM164, 20 µM zVAD-fmk for 0.5 h and then treated with 5 ng/mL mTNFα for 1 h. Representative images and enlarged nuclear staining of p-S166-RIPK1 are shown. Cell membranes were stained with Phalloidin (yellow). Nuclear membrane was stained with Lamin B (green). Nuclei were stained with Hoechst (blue). Quantification of the mean number of p-S166-RIPK1 puncta per nucleus in each field is shown on the right (n = 5). **P < 0.01, n.s., not significant. h Ripk1D325A/D325A MEFs were treated with or without 20 µM Nec-1s for 2 h followed by 20 ng/mL mTNFα for 1 h. The fraction of nucleus or cytoplasm was analyzed by western blotting. i Ripk1D325A/D325A;Ripk3−/− MEFs were treated with or without 20 µM Nec-1s for 2 h followed by 100 ng/mL mTNFα for 1 h or treated with 50 µM SM164, 20 µM zVAD-fmk for 0.5 h and then treated with 5 ng/mL mTNFα for 1 h. The fraction of nucleus or cytoplasm was analyzed by western blotting. Quantification of the ratio of nuclear/cytoplastic p-S166-RIPK1 or total RIPK1 were shown on the right. j Immunostaining of p-S166-RIPK1 in Ripk1D325A/D325A;Ripk3−/− MEFs treated with vehicle, or ± 20 µM Nec-1s for 0.5 h followed by 20 ng/mL mTNFα for 1 h. Representative images with enlarged nuclear staining of p-S166-RIPK1 and quantification of the mean number of p-S166-RIPK1 puncta per nucleus in each field are shown (n ≥ 8). *P < 0.05.