Fig. 4.
Fig. 4. In vitro binding studies with GST-Lyn fusion proteins. (A) Schematic representation of LynA and recombinant Lyn proteins used in in vitro binding studies. The SH3 and SH2 domains are represented by hatched and solid boxes, respectively. Amino acid numbers are shown under Lyn. (B) Cell lysate from parental 32D (EpoR −) or 32D/EpoR-Wt (EpoR +) cells, which were unstimulated or stimulated with Epo as indicated (Epo st. − or +, respectively), was incubated with GST-Lyn fusion proteins indicated below the panel. Affinity-purified proteins were resolved by SDS-PAGE and subjected to anti-EpoR immunoblotting. The tyrosine-phosphorylated 72-kD EpoR as well as the 62- to 66-kD forms of the EpoR is indicated. (C)Cell lysate from Epo-stimulated or unstimulated 32D/EpoR-Wt was incubated with GST (C) or GST-Lyn fusion proteins indicated below the panel. Affinity-purified proteins were subjected to antiphosphotyrosine immunoblotting followed by reprobing with anti-EpoR and anti-Jak2, as indicated. (D) 32D/EpoR-Wt cells were starved overnight and stimulated with 100 U/mL of Epo for 5 minutes or left unstimulated, as indicated, before solubilization. Cell lysates were immunoprecipitated with anti-EpoR or anti-Jak2 (as indicated), resolved by SDS-PAGE, and electrotransferred onto a PVDF membrane. The membrane was probed with GST-Lyn119-230 followed by detection with anti-GST immunoblotting. The membrane was then reprobed with GST-Lyn1-118, GST-Lyn231-512, GST (C), antiphosphotyrosine (αPY), and the antibody used for immunoprecipitation, as indicated. (E) Cell lysate from Epo-stimulated 32D/EpoR-Wt was mixed with synthetic phosphopeptides (200 μmol/L) corresponding to potential tyrosine phosphorylation sites, as indicated, and incubated with GST-Lyn119-230. Proteins bound to GST-Lyn119-230 were then subjected to immunoblotting with anti-EpoR. (F) Cell lysate from Epo-stimulated 32D/EpoR-Wt was mixed with indicated concentrations of the Y-464 and Y479 phosphopeptides, as indicated, or the unphosphorylated equivalent of Y-479 polypeptide (479DP) and analyzed as described above.

In vitro binding studies with GST-Lyn fusion proteins. (A) Schematic representation of LynA and recombinant Lyn proteins used in in vitro binding studies. The SH3 and SH2 domains are represented by hatched and solid boxes, respectively. Amino acid numbers are shown under Lyn. (B) Cell lysate from parental 32D (EpoR −) or 32D/EpoR-Wt (EpoR +) cells, which were unstimulated or stimulated with Epo as indicated (Epo st. − or +, respectively), was incubated with GST-Lyn fusion proteins indicated below the panel. Affinity-purified proteins were resolved by SDS-PAGE and subjected to anti-EpoR immunoblotting. The tyrosine-phosphorylated 72-kD EpoR as well as the 62- to 66-kD forms of the EpoR is indicated. (C)Cell lysate from Epo-stimulated or unstimulated 32D/EpoR-Wt was incubated with GST (C) or GST-Lyn fusion proteins indicated below the panel. Affinity-purified proteins were subjected to antiphosphotyrosine immunoblotting followed by reprobing with anti-EpoR and anti-Jak2, as indicated. (D) 32D/EpoR-Wt cells were starved overnight and stimulated with 100 U/mL of Epo for 5 minutes or left unstimulated, as indicated, before solubilization. Cell lysates were immunoprecipitated with anti-EpoR or anti-Jak2 (as indicated), resolved by SDS-PAGE, and electrotransferred onto a PVDF membrane. The membrane was probed with GST-Lyn119-230 followed by detection with anti-GST immunoblotting. The membrane was then reprobed with GST-Lyn1-118, GST-Lyn231-512, GST (C), antiphosphotyrosine (αPY), and the antibody used for immunoprecipitation, as indicated. (E) Cell lysate from Epo-stimulated 32D/EpoR-Wt was mixed with synthetic phosphopeptides (200 μmol/L) corresponding to potential tyrosine phosphorylation sites, as indicated, and incubated with GST-Lyn119-230. Proteins bound to GST-Lyn119-230 were then subjected to immunoblotting with anti-EpoR. (F) Cell lysate from Epo-stimulated 32D/EpoR-Wt was mixed with indicated concentrations of the Y-464 and Y479 phosphopeptides, as indicated, or the unphosphorylated equivalent of Y-479 polypeptide (479DP) and analyzed as described above.

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