Extended Data Fig. 7: SILAC experiments demonstrate that mutating IRF6 Ser413 and Ser424 to Ala reduces the amount of phosphorylation at IRF6 Ser90 by half.
From: The RIPK4–IRF6 signalling axis safeguards epidermal differentiation and barrier function
a, Schematic of the SILAC experiment that was performed to compare the amount of phosphorylation at S90, S413 and S424 between wild-type and mutant IRF6. b, Extracted ion chromatograms of the peptide phosphorylated at Ser90 (pSREFNLMoxYDGTK) and its unmodified counterpart (SREFNLMoxYDGTK), from wild-type IRF6 (light labelled) and IRF6(S413A/S424A) (heavy labelled). More phosphorylation at Ser90 was observed in wild-type IRF6 than in IRF6(S413A/S424A) (log2(S413A.S424A/WT) = −0.5) even though there was more total IRF6(S413A/S424A) (log2(S413A.S424A/WT) = 0.6). When normalized for total IRF6 levels, log2(S413A.S424A/WT) ≈ −1.1; that is, 2.1-fold less pS90 in IRF6(S413A/S424A) compared to wild-type IRF6. c, Extracted ion chromatograms of the peptide phosphorylated at Ser413 or Ser416 (pSFDSGSVR) and its unmodified counterpart (SFDSGSVR), from wild-type IRF6 (light labelled) and IRF6(S90A) (heavy labelled). When normalized for total IRF6 levels, log2(S90A/WT) = 0.11, so pS413 is at a similar level in wild-type IRF6 and IRF6(S90A). d, Extracted ion chromatograms of the peptide phosphorylated at Ser424 (long peptide: LQIpSTPDIKDNIVAQLK) and its unmodified counterpart (long peptide: LQISTPDIKDNIVAQLK), from wild-type IRF6 (light labelled) and IRF6(S90A) (heavy labelled). pS424 is at a similar level in wild-type IRF6 and IRF6(S90A) (log2(S90A/WT) = 0.2). e, Extracted ion chromatograms of the peptide phosphorylated at Ser90 (pSREFNLMoxYDGTK) and its unmodified counterpart (SREFNLMoxYDGTK), from IRF6(S423A/S424A) (light labelled) and IRF6(S413E/S424E) (heavy labelled). More pS90 was observed in IRF6(S413E/S424E) than in IRF6(S413A/S424A) (log2(S413E.S424E/S413A.S424A) = 0.6) even though there was slightly less total IRF6(S413E/S424E) (log2(S413E.S424E/S413A.S424A) = −0.4). When normalized for total IRF6 levels, log2(S413E.S424E/S413A.S424A) ≈ 1.0; that is, twofold less pS90 in IRF6(S413A/S424A) compared to IRF6(S413E/S424E). f, Extracted ion chromatograms of the peptide phosphorylated at Ser90 (pSREFNLMoxYDGTK) and its unmodified counterpart (SREFNLMoxYDGTK), from wild-type IRF6 (light labelled) and IRF6(S413E/S424E) (heavy labelled). More pS90 was observed in IRF6(S413E/S424E) than in wild-type IRF6 (log2(S413E.S424E/WT) = 0.7) even though there was also slightly more total IRF6(S413E/S424E) (log2(S413E.S424E/WT) = 0.1). When normalized for total IRF6 levels, log2(S413E.S424E/WT) ≈ 0.6; that is, 1.52-fold more pS90 in IRF6(S413E/S424E) compared to wild-type IRF6. Chromatograms are representative of two experiments. Notably, these are extracted ion chromatograms of representative peptide spectral matches (PSMs). The reported log2 values were calculated from all the different types of PSMs that cover the phosphorylation sites of interest.
