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. 2001 Aug 6;194(3):275–284. doi: 10.1084/jem.194.3.275

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© 2001 The Rockefeller University Press

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p62^dok controls MAPK and ras activation. (A and B) Phosphorylation of p62^dok (indicated by an asterisk) and ERK1/2 in response to PDGF. PEF were serum starved and then stimulated with 50 ng/ml PDGF for 10 min. Then, the cells were washed and serum-free medium was added to analyze the kinetics of ERK inactivation. The amount of cell extracts was normalized using an antibody recognizing ERK1/2 independently of its phosphorylation status. Normalized densitometric analysis of ERK1/2 phosphorylation is shown in B. (C) Ras GTP/GDP binding analysis. Wild-type and p62^dok−^/− PEFs were serum starved, labeled with ³²Pi for 18 h, and treated with 50 ng/ml PDGF for 10 min. Then the cells were washed twice and incubated in phosphate-free and serum-free medium for additional 15 and 30 min. Cells at different indicated time points were lysed, ras was immunoprecipitated, and the GTP and GDP present in the immunoprecipitates were resolved on a thin-layer polyethyleneimine-cellulose plate. The radioactivity in the GTP and GDP was quantitated with a Fuji Photo Film Co., BAS2000 PhosphorImager. The data are expressed as percent GTP, which was calculated by value_GTP /(1.5 × value_GDP + value_GTP) × 100. Wild-type, filled circle and dashed line; p62^dok−^/−, filled diamond and solid line (D and E). Phosphorylation of ERK1/2 in BMMCs in response to IL-3. BMMCs were serum starved and then stimulated with 2.5 ng/ml IL-3 for 5 min. Then, the cells were washed and serum-free medium was added to analyze the kinetics of ERK inactivation. The amount of cell extracts was normalized using an antibody recognizing ERK1/2 independently of its phosphorylation status. Normalized densitometric analysis of ERK1/2 phosphorylation is shown in E. All graphs are representative examples of experiments repeated three to five times.

p62^dok controls MAPK and ras activation. (A and B) Phosphorylation of p62^dok (indicated by an asterisk) and ERK1/2 in response to PDGF. PEF were serum starved and then stimulated with 50 ng/ml PDGF for 10 min. Then, the cells were washed and serum-free medium was added to analyze the kinetics of ERK inactivation. The amount of cell extracts was normalized using an antibody recognizing ERK1/2 independently of its phosphorylation status. Normalized densitometric analysis of ERK1/2 phosphorylation is shown in B. (C) Ras GTP/GDP binding analysis. Wild-type and p62^dok−^/− PEFs were serum starved, labeled with ³²Pi for 18 h, and treated with 50 ng/ml PDGF for 10 min. Then the cells were washed twice and incubated in phosphate-free and serum-free medium for additional 15 and 30 min. Cells at different indicated time points were lysed, ras was immunoprecipitated, and the GTP and GDP present in the immunoprecipitates were resolved on a thin-layer polyethyleneimine-cellulose plate. The radioactivity in the GTP and GDP was quantitated with a Fuji Photo Film Co., BAS2000 PhosphorImager. The data are expressed as percent GTP, which was calculated by value_GTP /(1.5 × value_GDP + value_GTP) × 100. Wild-type, filled circle and dashed line; p62^dok−^/−, filled diamond and solid line (D and E). Phosphorylation of ERK1/2 in BMMCs in response to IL-3. BMMCs were serum starved and then stimulated with 2.5 ng/ml IL-3 for 5 min. Then, the cells were washed and serum-free medium was added to analyze the kinetics of ERK inactivation. The amount of cell extracts was normalized using an antibody recognizing ERK1/2 independently of its phosphorylation status. Normalized densitometric analysis of ERK1/2 phosphorylation is shown in E. All graphs are representative examples of experiments repeated three to five times.