Effect of the p.Gly269Asp β1-tubulin variant in platelets and megakaryocytes. The functional and structural consequences of the novel p.Gly269Asp β1-tubulin variant were assessed in members of Pedigree H. (A) Immunofluorescence analysis of β1-tubulin was performed on poly-l-lysine–coated coverslips in platelets (platelet-rich plasma) from p.Gly269Asp carriers with (II.2 and III.3) or without thrombocytopenia (II.3) and a noncarrier (II.4). Platelets were labeled with a β1-tubulin antibody (red). Scale bars are 5 μm, and the objective lens is 63× (magnification 3×). (B) Western blot pictures of β1-tubulin levels in platelet lysates; β-actin was used as an internal control. (C) Representative images of CD34⁺ peripheral blood cell–derived MKs in a thrombocytopenic p.Gly269Asp carrier (II.2), noncarrier (II.4), and healthy control. (D) Representative images of spread MKs. MKs were labeled with β1-tubulin (green) and rhodamine-phalloidin (red). Nuclei were stained with DAPI (blue). The white arrow indicates a MKs with disorganized β1-tubulin. Images were acquired in a Leica SP8 confocal microscope with a 63× objective lens (magnification 1.5×). Scale bars are 5 μm. (E) Classification of MKs in family members and controls (as described in Figure 3; ^#P < .05 vs TCP carriers). (F) Microtubule length of CD34⁺ peripheral blood cell–derived MKs from controls and patients with p.Gly269Asp β1-tubulin variant was assessed using ImageJ software. Values are mean ± SD of values obtained from at least 10 different microscopy fields. TCP, thrombocytopenic; non-TCP, nonthrombocytopenic. **P < .005.