Improved chemotactic activity of cells pretreated with SDF-1 and TGF-β1 is associated with enhanced Erk1/Erk2 phosphorylation in response to SDF-1. (A) Ex vivo–expanded CD34⁺ cells were cultured in cytokine cocktail alone (medium) or along with TGF-β1, SDF-1, or SDF-1 and TGF-β1 for 24 hours. Cells were harvested, washed, and either left unstimulated or stimulated with 200 ng/mL SDF-1 for 3 minutes. Cells were solubilized, and extracts were subjected to immunoblotting by using anti–phospho Erk1/Erk2 or anti–phospho-Akt (Ser473) antibodies. After stripping and saturating nonspecific protein binding, we reprobed the same blots with anti–Erk1/Erk2 or anti-Akt antibody. Blots were developed by a chemiluminescence reaction and exposed to radiographic film. A representative experiment of 1 of 3 experiments performed is shown. (B) Effect of MEK inhibitor, PD98059, on migration activity of CD34⁺ cells pretreated with cytokine cocktail along with SDF-1 (200 ng/mL) or SDF-1 (200 ng/mL) + TGF-β1 (0.5 ng/mL) SDF-1 for 24 hours. Cells were tested for their migration response to SDF-1 (200 ng/mL). Data represent the mean ± SD of 4 independent experiments.