Fig 1: BD directly bound to ICAT. (A) Computer simulation of BD binding to ICAT, and a hydrogen bond was generated between BD and Lys 28 on ICAT. Immunoblot analysis of DARTS sample further revealed the increased stabilization of ICAT during the proteolysis process in HepG2 (B and D) and Huh7 cells (F and H). BD significantly increased the thermal stability of ICAT in CETSA assay at 49 °C and 52 °C in HepG2 (C and E) and Huh7 cells (G and I). (J) MST analysis of BD binding to wild type ICAT (Kd = 33.2 μmol/L). (K) MST analysis of BD binding to the mutant ICAT (Kd = 250 μmol/L). Data are shown as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus the control group.
Fig 2: Knockdown of ICAT increased the resistance to BD treatment, BD disrupted the interaction of ICAT and β-catenin. Repression of ICAT by siRNA significantly reversed the inhibition effect of BD on the HIF-1α and its mediated glucose metabolic pathway in HepG2 (A and C) and Huh7 cells (B and D). Relative glucose uptake in si-ICAT HCC cells treated with BD for 24 h (E). Relative lactate production in si-ICAT HCC cells with BD for 24 h (F and G). BD inhibited the expression of β-catenin under hypoxia in HCC cells (H and I). Co-IP assay revealed that BD disturb the interaction between β-catenin and ICAT (J and K). Data are shown as mean ± SD (n = 3). ∗ and #P < 0.05, ∗∗ and ##P < 0.01, ∗∗∗ and ###P < 0.001 versus the control group.
Supplier Page from Abcam for Recombinant Human CTNNBIP1/ICAT protein (His tag N-Terminus)