Extended Data Fig. 1: Domains of insulin receptor (IR) and activities of insulin analogs in primary mouse hepatocytes.
From: Synergistic activation of the insulin receptor via two distinct sites
a. Domains and disulfide connectivity of IR. L1 and L2, leucine rich domains 1 and 2; CR, cysteine rich domain; F1, F2 and F3, fibronectin III (FnIII) domains; ID, insert in FnIII-2 domain; TM, transmembrane domain; TK, tyrosine kinase domain. b. HPLC traces for each of the insulins synthesized and utilized for both functional and structural studies. c. MS1 spectra of the purified insulins in B analyzed in the Orbitrap mass analyzer. d. Binding of insulin WT and site−1 mutant (IleA2A;ValA3A) labeled with Alexa Fluor 488 to purified IR WT in the indicated conditions (Mean ± SD, WT, n = 9 independent experiments; IleA2A;ValA3A, n = 3). Significance calculated using two-tailed student t-test; **p < 0.01 and ***p < 0.001 (The exact p values are provided in the source data). e. Insulin competition-binding assay for isolated FnIII-1 domain and insulin WT and mutants (ValA3E and LeuB17R) (Mean ± SD, n = 3). f. Insulin-induced IR autophosphorylation in 293FT cells expressing IR wild-type (WT). Cells were treated with the indicated insulin WT or site-2 mutants for 10 min. The IR autophosphorylation levels were assessed by quantitative western blotting with a phospho-tyrosine (pY) IRβ antibody. Expression levels of IRβ were monitored by anti-Myc blotting against the C-terminal Myc-tag. g. Quantification of the western blot data shown in e (Mean ± SD). Each experiment was repeated four times. Significance calculated using two-tailed student t-test; *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001 (The exact p values are provided in the source data). Uncropped images for all blots and gels are available as source data.
