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Supplemental figure 2: A) Flow cytometry analysis showing that the TF1 erythroleukemia cells express high levels of CD34 marker on their cell surface. B) Twenty four hours post DOCK4 siRNA nucleofection, cells were used for immunoblot analysis for DOCK4 expression. Data are mean {plus minus}SEM from five independent experiments. C) Twenty four hours post DOCK4 knockdown, TF1 cells were briefly deprived of cytokines and then re-exposed to a cytokine cocktail (Stem cell factor (SCF), Interleukin-3 (IL-3) and Granulocyte-Macrophage colony stimulating factor (GM-CSF)) for 15 mins. Samples were resolved by immunoblotting for phospho-LYN (Y397), phospho-SHIP1 (Y1021) and phospho-SHP1 (Y536). Same membranes were probed with total LYN, SHIP1 and SHP1 antibodies as loading controls. D, E and F) Quantitation of the levels of phospho-LYN (Y397) (N=3), phospho-SHIP1 (Y1021) (N=5) and phospho-SHP1 (Y536) (N=4) respectively in TF1 cells from control and DOCK4 knocked down samples (*P < 0.05; Student's t test). Data are mean {plus minus}SEM from independent experiments as specified. G) Twenty four hours post DOCK4 knockdown HSCs were briefly deprived and exposed to 5 cytokine cocktails (TPO, SCF, FLT3 ligand, IL3 and IL6) for 15 mins. Following cytokine exposure, LYN kinase activity assay was performed to examine the endogeneous LYN kinase activity in HSCs expressing normal and reduced levels of DOCK4 (*P < 0.05; Student's t test). Data are represented as mean {plus minus}SEM from three technical replicates. H) Quantitation of the levels of phospho-SHIP1 (Y1021) in the in vitro kinase reaction products (***P < 0.0005; One way ANOVA). Data are mean {plus minus}SEM from three independent experiments. I) Quantitation of the levels of phospho-SHP1 (Y536) in the in vitro kinase reaction products (***P < 0.0005; One way ANOVA). Data are mean {plus minus}SEM from three independent experiments. J, K and L) Quantitation of the levels of phospho-LYN (Y397), phospho-SHIP1 (Y1021), and phospho-SHP1 (Y536) respectively in primary human HSCs treated with increasing doses of LYN/Src inhibitor, RK20449. Data are mean {plus minus}SEM from three biological replicates (****P < 0.00005, One way ANOVA; ns-not significant). M) LYN kinase activity assay was performed using recombinant LYN kinase and increasing amounts of recombinant DOCK4 C-terminus. JAK2 kinase and LYN/Src inhibitor were used as controls. Data are mean {plus minus}SEM from three independent experiments. (**P < 0.005; One way ANOVA). N) An in vitro binding assay was performed using flag-tagged DOCK4 C-terminus (D4CT) and recombinant LYN kinase to determine whether LYN kinase interacted with DOCK4-C-terminus. Samples were immunoprecipitated with anti-Flag antibody followed by immunoblot analysis using anti-Flag and anti-LYN antibodies. |
