The levels of nonphosphorylated Sp1 are increased by Mpl-ligand in Y10 megakaryocytes. In a Western blot of a 7% SDS-PAGE, lanes were loaded each with 10 μg nuclear extract prepared from Y10 cells treated for 3 days with or without 25 ng/mL MGDF in the absence or presence 100 nmol/L OA, as indicated. The major bands detected were 105 and 95 kD, as also reported by others.31,32 One arrow points to the major phosphorylated form of Sp1 (designated as Pi-Sp1 of ∼115 kD), and a second arrow points to the nonphosphorylated form of Sp1 (105 kD). The blot was treated with the ECL system as indicated in Materials and Methods. Equal loading of protein per lane was confirmed by staining with Ponceau S (not shown),24 followed by destaining and antibody reaction. The results shown are of a representative experiment out of three performed.