PDGFR activated by CLL-CM is upstream of Akt activation and is important for CLL-CM–driven MSC proliferation. (A) RTK array analysis of CLL MSC stimulated by CLL-CM for 30 minutes. The results are representative of more than 3 independent experiments. (B) PDGFRα and PDGFRβ phosphorylation were confirmed by immunoprecipitation of the CM-stimulated MSC lysates (incubation time, 30 minutes) with anti-PDGFRα or anti-PDGFRβ, followed by immunoblot analysis with antiphosphotyrosine antibody. After stripping the membrane, the upper 190-kDa protein coprecipitated with PDGFRα was confirmed to be PDGFRβ. The 170-kDa protein coprecipitated with PDGFRβ was found to be PDGFRα. The density of PDGFRβ phosphorylation and total protein expression was measured by densitometry analysis, and relative density was calculated by calculating the ratio of PDGFRβ phosphorylation to its total protein expression. The bar graph demonstrated the increased PDGFRβ phosphorylation when MSCs were exposed to CLL-CM. The results are representative of independent experiments (n = 3). CM, conditioned medium; pY, anti-phosphotyrosine antibody. (C) Both PDGFRα and Akt in CLL MSCs were phosphorylated by CLL-CM within 10 minutes of exposure. PDGFR tyrosine kinase inhibitor III (2μM) was able to block both PDGFRα and Akt phosphorylation. pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Two gels performed from one immunoprecipitation experiment were run and shown. Individual gels are shown in separate rectangle. (D) PDGFR inhibitor was able to partially block the proliferative effect of CLL-CM on MSCs. Six different CLL MSC sources were used in this experiment. (E) MSC proliferation increased in a dose-dependent manner to escalating doses of PDGF. Three different CLL MSCs were used in this experiment.