Fig. 1: PP6 is a previously unidentified component of complex I. | Cell Death & Disease

Fig. 1: PP6 is a previously unidentified component of complex I.

From: PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote TNFα-mediated cell death

Fig. 1

A MEFs were treated with Flag-TNFα or PBS for 15 min. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-Flag beads. The immunocomplexes were analyzed and quantified by mass spectrometry. The protein abundance represented as intensity was quantified by the summed peptide intensities of all extracted Ion chromatograms (XICs). Table below showed the proteins enriched in Flag-TNFα treatment group. B MEFs were treated with TNFα for indicated periods of time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-TNFR1 antibody. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. C PPP6R3+/+ or PPP6R3−/− MEFs were treated with PBS or TNFα for 30 min. In situ PLA assay was performed to detect the interaction between RIPK1 and PPP6R3. Red dots indicated the interaction signal of RIPK1 and PPP6R3. D Quantification analysis of the PLA signal dots of RIPK1 and PPP6R3. 20 cells were counted for each group. The panel on the right showed the knockout efficiency of PPP6R3. E PPP6R1, PPP6R2, or PPP6R3 knockout MEFs were treated with Flag-TNFα for indicated periods of time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-Flag beads. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. F WT or PPP6R1/2/3 triple-knockout MEFs were treated with Flag-TNFα for indicated periods of time. Cells were lysed with NP-40 buffer and immunoprecipitated with anti-Flag beads. The immunocomplexes and whole-cell lysates were analyzed by western blotting with indicated antibodies. Concentrations of reagents used: Flag-TNFα, 150 ng/ml; TNFα, 50 ng/ml.

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