Primary hematopoietic cells lacking L3MBTL1 show Epo-independent phosphorylation of STAT5, AKT, and MAP kinase. (A) Primary GFP⁺ CD34⁺ cells were harvested after one week in erythroid culture and lysed, according to standard protocols. Cultured CD34⁺ cells from MPN patients bearing the JAK2V617F mutation were harvested after 1 week of culture in Epo. Phosphorylation of STAT5, AKT, and MAPK was assessed by Western blot. The protein levels of JAK2, STAT5, AKT, MAPK, and L3MBTL1 were also assessed. (B) Primary GFP⁺ CD34⁺ cells were harvested after one week in erythroid culture and FOXO phosphorylation, and JNK expression levels were assessed by Western blot assay. Tubulin serves as a loading control for panels A, B, and E. (C) A total of 2 × 10⁷ GFP⁺ CD34⁺ cells, plated in erythroid culture conditions, were tested for Ras activation, by detection of GTP-bound Ras. Cells were lysed and incubated with (GST)-Raf-RBD fusion protein coupled to glutathione agarose beads. GTP-bound Ras was detected by Western blotting using an isoform-specific antibody. (D) Expression of the erythroid markers CD71 and GlyA was evaluated on L3MBTL1-KD CB cells cultured without Epo, in SCF, FLT-3, IL-6, and TPO. (E) After 1 week in culture without Epo, the GFP⁺ CD34⁺ cells were lysed, and phosphorylation of STAT5 and AKT was assessed by Western blot analysis. The protein levels of JAK2, STAT5, AKT, MAPK, Raf-1, p16, and L3MBTL1 were also assessed.