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. 2022 Jul 30;13:4434. doi: 10.1038/s41467-022-32214-2

Fig. 6. Release of CBP3 from the ternary complex after CLD3 proteolysis.

Fig. 6

a Fluorescence-detection size-exclusion chromatography (FSEC) of binary or ternary complexes after 10 h proteolysis by thrombin. The elution profile for standard IGF1-GFP (yellow), IGF1-GFP-strep/IGFBP3 binary complex (blue), and IGF1-GFP/IGFBP3/ALS-His ternary complex (red) are indicated as a control (top). b Immunoblot analysis of IGF1/myc-IGFBP3-Strep/ALS complex after 10 h proteolysis by thrombin and subsequent SEC. Anti-myc and anti-Strep antibody were used to detect NBP3 and CBP3, respectively. No-thrombin treatment served as negative control (Ctrl). c FSEC of the ternary complex (IGF1/IGFBP3-GFP/ALS-His) after thrombin digestion for indicated time. Fluorescence signal (GFP) was monitored to examine the dissociation of CBP3 from the ternary complex. d Quantification of immunoblot analysis for IGF1R phosphorylation after treatment of intermediate ternary complex. HEK293A cells were treated with IGF1-His (5 nM, positive control), IGF1-His/IGFBP3/ALS ternary complex (5 nM, none), or intermediate ternary complex (5 nM). The intermediate ternary complex was prepared by protease digestion (thrombin, ADAM12 and PAPP-A2) and subsequent SEC purification. No treatment was used for negative control (Ctrl). Data from three independent experiments (n  =  3) were analyzed and relative pIGF1R/IGF1R (Fold to Ctrl) were expressed as mean ± SD (ns, not significant vs. ctrl; ****P < 0.0001 vs. ctrl; ####P  <  0.0001 vs. none). P-values by one-way ANOVA test followed by Sidak’s multiple comparisons test. e SDS-PAGE analysis of the IGF1/hybrid-BPs/ALS ternary complex after thrombin digestion. f Quantification of immunoblot analysis for IGF1R phosphorylation with intermediate ternary complex containing hybrid-BPs. IGF1-His (5 nM, positive control), IGF1-His/IGFBP3/ALS ternary complexes (5 nM), or IGF1-His/hybrid-BPs/ALS ternary complexes (5 nM) with and without thrombin digestion were treated onto HEK293A cells. No treatment for negative control (Ctrl). Data from five independent experiments (n  =  5) were analyzed and relative pIGF1R/IGF1R (Fold to Ctrl) were expressed as mean ± SD (ns, not significant; ****P  <  0.0001 Ctrl vs. IGF1-His or -thrombin vs. +thrombin for each group). P-values by one-way ANOVA test followed by Sidak’s multiple comparisons test. g, h FSEC of the ternary complex IGF1-GFP/hybrid-BPs/ALS-His (g), or IGF1/hybrid-BPs-GFP/ALS-His (h) after thrombin digestion. Fluorescence signal (GFP) was monitored to examine the dissociation of IGF1 or CBP3 from the indicated ternary complex.