Fig. 2. MT-2 relaxes ASMCs specifically via transgelin-2.

(A) Confocal images of rat ASMCs showing the cellular membrane [DiIC₁₈(3), red], nucleus (Hoechst, blue), and MT-2 [FITC (fluorescein isothiocyanate), green] (scale bar, 10.0 μm). (B) Scatchard analysis of the binding of MT-2 to rat ASMCs (n = 3 per concentration). CPM, counts per minute. (C) Gel and Western blots using anti-His or anti–MT-2 antibody of immunoprecipitates of rat ASMCs lysates incubated with nickel–nitrilotriacetic acid beads linked to 6×His tag MT-2 recombinant protein used as a probe. (D) The competitive binding assays were performed with nonlabeled cold MT-2 (MT) or blocking antibodies against transgelin-2 (TG2), enolase-1 (ENO), D2 subclass of dopamine receptor (D2DR), or bovine serum albumin (BSA), whereas ¹²⁵I-MT-2 was used to quantify the specific binding of MT-2 to rat ASMCs (*P < 0.05 versus control without treatment, n = 3 per group). (E) Western blots of TG2 and β-actin of rat ASMCs treated with control or TG2 small interfering RNA (siRNA). Lower panel: Scatchard analysis of the binding of MT-2 to TG2-deficient rat ASMCs (n = 3 per concentration). (F) Confocal images of rat ASMCs showing TG2 (TG2, Cy3; red), nucleus (Hoechst, blue), and MT-2 (FITC, green) (scale bar, 10.0 μm). (G) Gel of purified rat recombinant TG2 protein and molecular weight markers (M). (H) Kinetic analysis of MT-2 by Biacore surface plasmon resonance with TG2 immobilized on the CM5 sensor chip. RU, response units. (I) Kinetic analysis of TG2 in Biacore surface plasmon resonance with MT-2 immobilized on the CM5 sensor chip. The response signals were recorded over the indicated time periods. All data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with the Games-Howell/LSD post hoc test.