Fig. 2.
Fig. 2. 4N1-1 and GPVI activation induce a similar tyrosine phosphorylation pattern. / Human platelets, preincubated as described in the legend for Figure 1with apyrase, indomethacin, and EGTA, were stimulated with 100 μM 4N1-1 from 10 to 240 seconds or for 2 minutes with 1 U/mL thrombin (Thr) or 10 μg/mL convulxin (Cvx). Platelets were then lysed by addition of NP-40 buffer. (A) Proteins of whole-cell lysate were resolved by 12.5% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. Arrows on the right indicate the position of prominent phosphorylated bands. (B) Cell extracts were immunoprecipitated by using the anti-Syk antibody. Immunoprecipitates were resolved by 10% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (top). The filter was dehybridized and reprobed by using the anti-Syk antibody (bottom). (C-E) Human platelets were stimulated with 100 μM 4N1-1 or 10 μg/mL convulxin and lysed 2 minutes later. (C) Cell extracts were immunoprecipitated by using the anti-PLCγ2 antibody. Immunoprecipitates were resolved by 7% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (top). The filter was dehybridized and reprobed by using the anti-PLCγ2 antibody (bottom). (D) Cell extracts were immunoprecipitated by using the anti–SLP-76 antibody. Immunoprecipitates were resolved by 10% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (top). The filter was dehybridized and reprobed by using the anti–SLP-76 antibody (bottom). (E) Cell extracts were precipitated by using GST fusion protein containing the tandem SH2 domain of Syk. Precipitated proteins were resolved by 15% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. (F) Cell extracts were precipitated by using convulxin and an anticonvulxin antibody (IP GPVI), the anticonvulxin antibody alone (Ab), or the GST-SH2-Syk fusion protein. Precipitated proteins were resolved by 15% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. Coll indicates collagen.

4N1-1 and GPVI activation induce a similar tyrosine phosphorylation pattern.

Human platelets, preincubated as described in the legend for Figure 1with apyrase, indomethacin, and EGTA, were stimulated with 100 μM 4N1-1 from 10 to 240 seconds or for 2 minutes with 1 U/mL thrombin (Thr) or 10 μg/mL convulxin (Cvx). Platelets were then lysed by addition of NP-40 buffer. (A) Proteins of whole-cell lysate were resolved by 12.5% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. Arrows on the right indicate the position of prominent phosphorylated bands. (B) Cell extracts were immunoprecipitated by using the anti-Syk antibody. Immunoprecipitates were resolved by 10% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (top). The filter was dehybridized and reprobed by using the anti-Syk antibody (bottom). (C-E) Human platelets were stimulated with 100 μM 4N1-1 or 10 μg/mL convulxin and lysed 2 minutes later. (C) Cell extracts were immunoprecipitated by using the anti-PLCγ2 antibody. Immunoprecipitates were resolved by 7% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (top). The filter was dehybridized and reprobed by using the anti-PLCγ2 antibody (bottom). (D) Cell extracts were immunoprecipitated by using the anti–SLP-76 antibody. Immunoprecipitates were resolved by 10% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (top). The filter was dehybridized and reprobed by using the anti–SLP-76 antibody (bottom). (E) Cell extracts were precipitated by using GST fusion protein containing the tandem SH2 domain of Syk. Precipitated proteins were resolved by 15% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. (F) Cell extracts were precipitated by using convulxin and an anticonvulxin antibody (IP GPVI), the anticonvulxin antibody alone (Ab), or the GST-SH2-Syk fusion protein. Precipitated proteins were resolved by 15% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. Coll indicates collagen.

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