|
|
Blood, Vol. 104, Issue 13, 3901-3910, December 15, 2004
Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1
Blood Harder et al.
104: 3901
Supplemental materials for: Harder et al, Vol 104, Issue 13, 3901-3910
Files in this Data Supplement:
- Figure S1. Lyn kinase is not a critical signaling component in erythroblast EPO-dependent activation of the Jak/STAT/MAP kinase pathways (JPG, 160 KB)
-
(A) CD71+Ter119+ erythroblasts were purified from Lyn+/+ and Lyn-/- spleens following treatment of the mice with phenylhydrazine using an anti-CD71 mAb and immunomagnetic bead purification. A representative example of pre- and postpurified spleen cells is shown. (B) Upper and lower panels show May-Grunwald Giemsa–stained cytospin preparations of purified Lyn+/+ and Lyn-/- cells. (C) Purified cells from Lyn+/+ mice were stimulated with 0, 10 or 100 U/mL of EPO for 5 minutes before lysis. The EPO receptor or Jak2 were then precipitated and assessed for phosphotyrosine content with anti-PY antibodies (Abs). Blots were probed with either anti-EPOr or anti-Jak2 Abs to ensure equal loading. Precipitates with normal rabbit serum were conducted as controls (NRS). Alternatively, total cell lysates were blotted with the indicated Abs to investigate activation of STAT5 and the MAP kinases Erk1 and Erk2. (D) CD71+Ter119+ cells were purified as described in panel A from Lyn+/+ and Lyn-/- mice, and activation of STAT5 and MAP kinases was assessed following stimulation of cells with the indicated doses of EPO for 5 minutes or with 100 U/mL EPO for the indicated amounts of time. (E) SHIP-1 was immunoprecipitated from Lyn+/+ and Lyn-/- erythroblasts and its level of tyrosine-phosphorylation determined by anti-PY blotting. Precipitates were then reprobed with anti–SHIP-1 Abs to ensure that equal amounts of SHIP-1 were precipitated. Control precipitations are also shown (NRS).
|
|
