Fig. 4.
Fig. 4. Effect of EPO and SCF on tyrosine protein phosphorylation in HCD57 cell and HCD57-SREI cells. (A) HCD57 cells deprived of EPO overnight (lanes A, B, C) or HCD57-SREI cells cultured without EPO (lanes D, E, F) were left untreated (A, D) or treated with 10 U EPO/mL for 10 minutes (B, E) or 100 ng SCF/mL for 10 minutes (C, F). The cells were then rapidly chilled in an ice bath, a detergent lysate was prepared, and phosphotyrosine containing proteins were immunoprecipitated with a polyclonal anti-Tyr(P) antibody. The immunoprecipitated proteins were then run on SDS-polyacrylamide gel electrophoresis, Western blotted, and probed with a monoclonal anti-Tyr(P) antibody (top panel) and then stripped of bound antibody and reprobed with an anti-JAK2 antiserum (bottom panel). Bands were visualized with ECL. (B) SHC protein tyrosine phosphorylation in HCD57-SREI cells (lanes 1 through 3) and HCD57 cells (lanes 4 through 6). The cells were treated with nothing (lanes 1 and 4), SCF (lanes 2 and 5), and EPO (lanes 3 and 6) as described above and SHC proteins were immunoprecipitated. The Western blot was probed with anti-Tyr(P) (top panel) and then stripped of bound antibody and reprobed with anti-SHC antiserum (bottom panel). (C) MAP kinase (ERK-1) activity in HCD57 and HCD57-SREI cells. HCD57 cells were deprived of EPO overnight in IMDM containing 25% serum. Aliquots of HCD57 cells that had been deprived of EPO (lanes 1 through 4) and HCD57-SREI cell continually cultured without EPO (lanes 5 through 8) were deprived of serum for 1 hour. After this 1-hour incubation in serumless medium, either nothing (lanes 1 and 5), 1.0 U EPO/mL (lanes 2 and 6), 25% fetal calf serum (lanes 3 and 7), or a combination of 25% serum plus 1.0 U EPO/mL was added for 10 minutes at 37°C. After the 10-minute incubation, the cells were rapidly chilled in cold medium, solubilized, and the ERK-1, MAP kinase activity determined by immunoprecipitation and phosphorylation of myelin basic protein as described in Materials and Methods. (D) Constitutive STAT5 phosphorylation in HCD57-SREI cells. HCD57-SREI cells (lanes A through C, G through I) and HCD57 cells (lanes E through F, J through L) were treated with nothing (lanes A, D, G, J) or 10 U EPO/mL for either 5 minutes (lanes B, E, H, K) or 10 minutes (lanes C, F, I, L) at 37°C. Ten micrograms of total cellular extract (lanes A through F) or STAT5 immunoprecipitated from 200 μg of cellular extract (lanes G through L) were analyzed by Western blotting with anti-Tyr(P) antiserum. Molecular-weight markers and migration of proteins of interest are indicated.

Effect of EPO and SCF on tyrosine protein phosphorylation in HCD57 cell and HCD57-SREI cells. (A) HCD57 cells deprived of EPO overnight (lanes A, B, C) or HCD57-SREI cells cultured without EPO (lanes D, E, F) were left untreated (A, D) or treated with 10 U EPO/mL for 10 minutes (B, E) or 100 ng SCF/mL for 10 minutes (C, F). The cells were then rapidly chilled in an ice bath, a detergent lysate was prepared, and phosphotyrosine containing proteins were immunoprecipitated with a polyclonal anti-Tyr(P) antibody. The immunoprecipitated proteins were then run on SDS-polyacrylamide gel electrophoresis, Western blotted, and probed with a monoclonal anti-Tyr(P) antibody (top panel) and then stripped of bound antibody and reprobed with an anti-JAK2 antiserum (bottom panel). Bands were visualized with ECL. (B) SHC protein tyrosine phosphorylation in HCD57-SREI cells (lanes 1 through 3) and HCD57 cells (lanes 4 through 6). The cells were treated with nothing (lanes 1 and 4), SCF (lanes 2 and 5), and EPO (lanes 3 and 6) as described above and SHC proteins were immunoprecipitated. The Western blot was probed with anti-Tyr(P) (top panel) and then stripped of bound antibody and reprobed with anti-SHC antiserum (bottom panel). (C) MAP kinase (ERK-1) activity in HCD57 and HCD57-SREI cells. HCD57 cells were deprived of EPO overnight in IMDM containing 25% serum. Aliquots of HCD57 cells that had been deprived of EPO (lanes 1 through 4) and HCD57-SREI cell continually cultured without EPO (lanes 5 through 8) were deprived of serum for 1 hour. After this 1-hour incubation in serumless medium, either nothing (lanes 1 and 5), 1.0 U EPO/mL (lanes 2 and 6), 25% fetal calf serum (lanes 3 and 7), or a combination of 25% serum plus 1.0 U EPO/mL was added for 10 minutes at 37°C. After the 10-minute incubation, the cells were rapidly chilled in cold medium, solubilized, and the ERK-1, MAP kinase activity determined by immunoprecipitation and phosphorylation of myelin basic protein as described in Materials and Methods. (D) Constitutive STAT5 phosphorylation in HCD57-SREI cells. HCD57-SREI cells (lanes A through C, G through I) and HCD57 cells (lanes E through F, J through L) were treated with nothing (lanes A, D, G, J) or 10 U EPO/mL for either 5 minutes (lanes B, E, H, K) or 10 minutes (lanes C, F, I, L) at 37°C. Ten micrograms of total cellular extract (lanes A through F) or STAT5 immunoprecipitated from 200 μg of cellular extract (lanes G through L) were analyzed by Western blotting with anti-Tyr(P) antiserum. Molecular-weight markers and migration of proteins of interest are indicated.

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