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. 2015 Jul 6;10(7):e0131866. doi: 10.1371/journal.pone.0131866

Fig 1. dnMpl does not transmit Thpo induced signals.

Fig 1

(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.