Flow cytometry, RT-PCR, and ligand blotting of GPVI in HEL and CMK cells.

(A) HEL and CMK cells were differentiated with PMA for 3 days and expression of GPVI detected by flow cytometry as in Figure 1. GPIIIa expression and ploidy values were measured with anti-GPIIIa and propidium iodide, respectively. Arrowheads in ploidy panels indicate DNA content, from 2n to 16n. (B) Semiquantitative RT-PCR of HEL and CMK cells shows expression of GPVI, glycophoryn A, GPIIIa (CD61), and HPRT before (lane 1) and after megakaryocytic differentiation with PMA for 1 (lane 2) and 3 days (lane 3) or erythroid differentiation using hemin for 3 days (lane 4). (C) Cells were differentiated with 10 nmol/L PMA for 1 and 3 days (1d, 3d). HEL extract (30 μg) was loaded per lane and subjected to SDS-PAGE under nonreducing conditions. GPVI was detected by ligand blotting using convulxin. FcRγ-chain expression was assessed by immunoblotting. Arrows indicate the relative position of GPVI (60 kd) and FcR γ-chain (22-24 kd). A platelet sample is included as a positive control; the platelet number used in either blot was different. The level of Syk measured by Western blotting is also shown for comparison. Results are representative of 3 experiments.