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. Author manuscript; available in PMC: 2024 Dec 21.
Published in final edited form as: Cell. 2023 Dec 5;186(26):5812–5825.e21. doi: 10.1016/j.cell.2023.11.009

Figure 5. S-nitrosylation of INSRβ by SNO-CoA-dependent SCAN activity regulates insulin signaling.

Figure 5.

(A) SNO-INSRβ and SNO-IRS1 in L6-SCAN-WT, L6-SCAN-QTG/NAA and L6-C109/188R cell lines, respectively. (B) Quantification of SNO-INSRβ and SNO-IRS1 in A; n=3. (C) Phosphorylation of IRS1(pTyr608) and AKT(pSer473) in overnight serum-starved L6-SCAN-WT, L6-SCAN-QTG/NAA and L6-C109/188R cell lines, 30 min after a 10-minute insulin treatment (100 nM). (D) Quantification of phosphorylation level of IRS1 and AKT in C; n=3. (E) Four peptides containing single candidate SNO sites (cysteine residues, red) within INSR identified by SNO-RAC-coupled mass spectroscopy. (F) Identification of primary SNO site within INSRβ. SNO-INSRβ with the indicated mutations of candidate SNO sites expressed in HEK cells. (G) Quantification of SNO-INSRβ in F; n=3. (H) Phosphorylation of AKT(pSer473) and AS160(pThr642) in overnight serum-starved INSR-WT and INSR-C1083A expressing L6 cells at 1, 30, 60, 120 and 240 min after removal of insulin, following a 10-minute insulin treatment (100 nM). (I-J) Quantification of phosphorylation of AKT(pSer473) (I) and AS160(pThr642) (J) in H, respectively; n=3. All results are presented as mean ± SD. Two-tailed Student’s t-test was used to detect significance in Fig. 5B and 5D. One-way ANOVA with Tukey post hoc was used to detect significance in Fig. 5G, 5I and 5J. *, p<0.05 and **, p<0.01. See also Figure S6.