Expression of specific PDGFRA mutations in 32D cells overcomes IL-3 dependence. 32D cells were retrovirally transduced with PDGFRA vectors containing mutations as specified in the results section or empty vector, FIP1L1-PDGFRA, JAK2wt, JAK2V617F, BCR-ABL1 or FLT3-ITD. (A) Growth capacity of all mutant PDGFRA cell lines without IL-3. PDGFRA H650Q, N659S, R748G, and Y849S induce IL-3–independent growth in 32D cells. Cells expressing the indicated mutations were seeded without IL-3 and counted daily for 5 days. The graph depicts mean viable cell numbers taken from a representative experiment performed in triplicates. The results were confirmed in 3 independent experiments. (B) Clonogenic growth in semisolid medium. The 32D cells expressing the indicated mutations were plated in triplicates in methyl cellulose media without IL-3 at a concentration of 200 cells per dish. Colonies were counted on day 6. These results were confirmed in 3 independent experiments. (C) Western blot experiments of 32D cell lines. Total cell lysates of IL-3– and 0.5% FCS-deprived (12-hour starvation) 32D cells were conducted. Blots were stained with the indicated phosphorylation-specific or total antibodies. PDGFRA and pPDGFRA antibodies recognize 2 bands, the mature form of the receptor with a size of approximately 180 kDa and an incompletely glycosylated precursor at about 160 kDa.³⁷  Stat5 and Stat5 phosphorylation were detected on a second membrane using same lysates and same amounts of protein.