Fig. 4: Knockdown of zyxin results in lysosome-mediated GPIbα degradation.

A mRNA levels of GPIbα, GPIbβ, and GPIX in WT and Zyx^−/− platelets. mRNA expression was analyzed by qRT-PCR and determined by a ratio relative to the control GAPDH. The data were expressed as the ratio relative to WT; n = 5 mice per genotype. B Western blot analysis of GPIbα protein in WT and Zyx^−/− platelets. Protein concentration was adjusted to the same level between WT and Zyx^−/− platelet lysates. Blots are representative of five independent experiments. C, D Dami cells were transfected with siRNAs targeting zyxin (siZYX-1, and -2) or negative control siRNA (control). The expression of GPIbα and GPIX in Dami cells was analyzed by Western blot; the blots are representative of five independent experiments (C). The surface level of GPIbα and GPIX was analyzed by flow cytometry (D). E, F Dami cells were treated with or without 10 μg/mL leupeptin plus 10 mM NH₄Cl (Leu + NH₄Cl) and MG-132 (100 nM) for 12 h after zyxin siRNA (siZYX-1) transfection. The total expression of GPIbα was analyzed with Western blot; the blots are representative of five independent experiments (E). The surface level of GPIbα was analyzed by flow cytometry (F). Data are from five independent experiments in (C–F). Means are indicated by horizontal lines in (A) and (C–F). *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA followed by Dunnett’s post hoc test in (C–F). NS, not significant.