Fig. 6.
Fig. 6. EPO-R and JAK2 tyrosine phosphorylation in 32D cells containing variants of the EPO-R. (A) Cells were washed in RPMI and placed in OptiMEM (GIBCO) in the absence of serum and growth factors. After 6 hours, cells were stimulated with 50 U/mL EPO (+) for 7 minutes or were not stimulated (−). Cell extracts from 7.5 × 105 cells were separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies. Lanes 1 and 2, 32D.EPO-R(1-483), wild-type cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells. Molecular mass standards (in kilodaltons) are on the left. (B) Cell extracts from starved cells (0 EPO) or cells stimulated with 1 U/mL EPO or 50 U/mL EPO were immunoprecipitated with antisera against the N-terminal peptide of the murine EPO-R. Bound products were washed and separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies (upper panel). The blot was then stripped and reprobed with antisera against the EPO-R (lower panel); arrowheads on the left indicate the position of wild-type (wt) EPO-R, EPO-R(W282R) and EPO-R(YF), IgG, and EPO-R(1-373), respectively. Lanes 1 through 3, 32D.EPO-R(1-483) wt cells; lanes 4 through 6, 32D.EPO-R(1-373) cells; lanes 7 and 8, 32D.EPO-R(YF) cells; and lanes 9 and 10, 32D.EPO-R(W282R) cells. (C) Cell extracts from starved cells (−) or cells stimulated with 50 U/mL EPO (+) were immunoprecipitated with antisera against JAK2. Bound products separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies (upper panel); the arrowhead on the left identifies the position of JAK2. The blot was then stripped and reprobed with antisera against JAK2 (lower panel). Lanes 1 and 2, 32D.EPO-R(1-483) wt cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells.

EPO-R and JAK2 tyrosine phosphorylation in 32D cells containing variants of the EPO-R. (A) Cells were washed in RPMI and placed in OptiMEM (GIBCO) in the absence of serum and growth factors. After 6 hours, cells were stimulated with 50 U/mL EPO (+) for 7 minutes or were not stimulated (−). Cell extracts from 7.5 × 105 cells were separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies. Lanes 1 and 2, 32D.EPO-R(1-483), wild-type cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells. Molecular mass standards (in kilodaltons) are on the left. (B) Cell extracts from starved cells (0 EPO) or cells stimulated with 1 U/mL EPO or 50 U/mL EPO were immunoprecipitated with antisera against the N-terminal peptide of the murine EPO-R. Bound products were washed and separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies (upper panel). The blot was then stripped and reprobed with antisera against the EPO-R (lower panel); arrowheads on the left indicate the position of wild-type (wt) EPO-R, EPO-R(W282R) and EPO-R(YF), IgG, and EPO-R(1-373), respectively. Lanes 1 through 3, 32D.EPO-R(1-483) wt cells; lanes 4 through 6, 32D.EPO-R(1-373) cells; lanes 7 and 8, 32D.EPO-R(YF) cells; and lanes 9 and 10, 32D.EPO-R(W282R) cells. (C) Cell extracts from starved cells (−) or cells stimulated with 50 U/mL EPO (+) were immunoprecipitated with antisera against JAK2. Bound products separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies (upper panel); the arrowhead on the left identifies the position of JAK2. The blot was then stripped and reprobed with antisera against JAK2 (lower panel). Lanes 1 and 2, 32D.EPO-R(1-483) wt cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells.

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