MLC2 phosphorylation status during megakaryocyte differentiation. (A) Immunolabeling was performed with an anti-pMLC2 (red), an anti-VWF (green), and a TOTO-3 iodide (blue). Photomicrographs were taken using a Zeiss laser scanning microscope equipped with a 63×/1.4 numerical aperture (NA) oil objective. pMLC2 was localized in the cytoplasm of immature (i-ii) and mature MKs (iii-iv). In MK-forming PPTs (v-vi), pMLC2 was localized along the cytoplasmic extensions (arrow 1), in the swellings (arrows 4 and 5), and in the tips of PPTs (arrows 2 and 3). Bars represent 5 μm. (B) CD34⁺ cells derived from leukapheresis were cultured 3 days with TPO, and one part of the cells was collected. Culture was continued, and CD41⁺ MKs were selected at day 5 as described in “Materials and methods” and grown for additional days with TPO. Western blots were performed on cells collected on day 3 and on MKs collected on days 6, 9, and 12. β-Actin, MLC2, and pMLC2 protein levels increased from day 3 to day 12. Rho was expressed at an almost constant level regardless of the MK differentiation stages. Anti-Hsc70 antibody was used as a control of protein loading. (C-D) Western blot quantification showed the increase in both pMLC2 (C) and pMLC2/MLC2 ratio (D) during MK differentiation.