Journal: Cell Death & Disease

Article Title: Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

doi: 10.1038/s41419-021-04246-x

Figure Lengend Snippet: A Differential proteomic analysis of proteins expressed in WT and Zyx −/− platelets; n = 3 mice per genotype. B Surface level of GPIb-IX complex on WT and Zyx −/− platelets analyzed by flow cytometry; n = 3 mice per genotype. MFI, mean fluorescence intensity. C Western blot analysis of GPIb-IX in WT and Zyx −/− platelets. Protein concentration has been adjusted to the same level between WT and Zyx −/− platelet lysates. Blots are representative of five independent experiments. D Expression of GPIb-IX complex in WT and Zyx −/− MKs. Left panels, representative confocal images of MKs in WT and Zyx −/− mouse femoral BM sections immunostained with anti-GPIbα, GPIbβ, and GPIX antibodies (original magnification ×200). GPIbα, GPIbβ, and GPIX in MKs are in green, and nuclei are in blue. Scale bar: 100 μm. Right panels, the quantification of MFI of GPIbα, GPIbβ, and GPIX in the left panels analyzed by ImageJ software; n = 10 visual fields from 5 mice per genotype. Data are expressed as means ± SD. E Percentage of GPIbα + cells differentiated from WT and Zyx −/− FL HPCs was analyzed by flow cytometry. F Percentage of GPIbα + platelet-sized particles released from culture-derived MKs in ( E ) was analyzed by flow cytometry. Data are from four independent experiments in ( E ) and ( F ). Means are indicated by horizontal lines in ( A ), ( B ), ( E ), and ( F ). ** P < 0.01, *** P < 0.001, compared with WT mice by unpaired Student’s t -test in ( A ), ( B ), and ( D ), and by two-way ANOVA followed by Bonferroni’s post hoc test in ( E ) and ( F ).

Article Snippet: Antibodies against zyxin (10330-1-AP), GPIX (14-564-1-AP), GFP tag (50430-2-AP), and flag tag (20543-1-AP) were from Proteintech (Wuhan, China).

Techniques: Flow Cytometry, Fluorescence, Western Blot, Protein Concentration, Expressing, Software, Derivative Assay

Journal: Cell Death & Disease

Article Title: Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

doi: 10.1038/s41419-021-04246-x

Figure Lengend Snippet: 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 (si ZYX -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 NH 4 Cl (Leu + NH 4 Cl) and MG-132 (100 nM) for 12 h after zyxin siRNA (si ZYX -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.

Article Snippet: Antibodies against zyxin (10330-1-AP), GPIX (14-564-1-AP), GFP tag (50430-2-AP), and flag tag (20543-1-AP) were from Proteintech (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Protein Concentration, Transfection, Negative Control, Flow Cytometry