Fig. 3: Loss of tyrosine phosphorylation of RIPK1 promotes TNF-induced apoptosis and necroptosis.
From: Tyrosine phosphorylation regulates RIPK1 activity to limit cell death and inflammation
a Primary WT BMDMs were stimulated by TNF with or without Nec-1 (RIPK1 inhibitor), JAK1 and SRC inhibitors for 24 h, and cell death were measured by SytoxGreen positivity. b, c Primary Ripk1+/+ and Ripk1Y383F/Y383F BMDMs were stimulated by TNF with or without Nec-1 (RIPK1 inhibitor), JAK1 and SRC inhibitors for 24 h. Cell lysates were collected for western blotting (c) and cell death were measured by SytoxGreen positivity (b). d Primary Ripk1+/+ and Ripk1Y383F/Y383F BMDMs were treated by different stimulators for 6 h. Cell death were measured by SytoxGreen positivity. T: TNF; B: BV-6; Z: zVAD.fmk; N: Necrostatin-1. Primary Ripk1+/+ and Ripk1Y383F/Y383F BMDMs were stimulated by TNF/BV-6 (e) and TNF/BV-6/zVAD (f) for indicated time points and whole-cell lysates were collected for western blotting. Ripk1+/+ and Ripk1Y383F/Y383F immortalized MEFs were stimulated with TNF/BV-6 (g) or TNF/BV-6/zVAD (h) for indicated time points and whole-cell lysates were immunoprecipitated using anti-RIPK1 (g) or anti-RIPK3 (h) antibody. TNF: 100 ng/ml (a–c) and 10 ng/ml (d–h); BV-6: 2.5 uM; zVAD.fmk: 20 μM; Necrostatin-1: 10 μM; JAK1 inhibitor: 10 μM; SRC inhibitor: 10 μM. In a, b, d, data are represented as mean ± SEM (n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test. n.s. p > 0.05; ****p < 0.0001. Source data are provided with this paper.
