The ligand for c-mpl, thrombopoietin, was cloned in 1994. Thrombopoietin was shown to be the major regulator of megakaryocytopoiesis and platelet formation. The protein encoded by the c-mpl gene, CD110, is a 635 amino acid transmembrane domain, with two extracellular cytokine receptor domains and two intracellular cytokine receptor box motifs. TPO-R deficient mice were severely thrombocytopenic, emphasizing the important role of CD110 and thrombopoietin in megakaryocyte and platelet formation. Upon binding of thrombopoietin, CD110 is dimerized and the JAK family of non-receptor tyrosine kinases, as well as the STAT family, the MAPK family, the adaptor protein Shc and the receptors themselves become tyrosine phosphorylated.
Background References
1. Jiang X et al. Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings. Phytomedicine. 2023 Feb
2. Guglielmelli P et al. The MPL mutation. Int Rev Cell Mol Biol. 2021
Western blot analysis of TPOR on different lysates with Rabbit anti-TPOR antibody (HA722068) at 1/1,000 dilution.
Lane 1: Mouse placenta tissue lysate (40 µg/Lane) Lane 2: NIH/3T3 cell lysate (20 µg/Lane) Lane 3: A431 cell lysate (no heat) (20 µg/Lane) Lane 4: Rat spleen tissue lysate (40 µg/Lane) Lane 5: Mouse spleen tissue lysate (40 µg/Lane)
Notice: no heat means the lysate is not boiled.
Predicted band size: 71 kDa Observed band size: 80 kDa
Exposure time: Lane 1-3: 24 seconds; Lane 4-5: 30 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722068) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Flow cytometric analysis of A431 cells labeling TPOR.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA722068, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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