FIG 5.

Syk phosphorylates nucleolin and promotes its binding to Bcl-x_L mRNA. (A) Tet-responsive MDA-MB-231 cells pretreated without or with doxycycline to induce Syk-EGFP were treated without or with 5 mM H₂O₂ for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (WCL; bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (B) DG75 B cells were pretreated with 50 μM piceatannol (PIC; +) or dimethyl sulfoxide carrier alone (−) and then treated without or with 5 mM H₂O₂ for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (C) DG75 cells were pretreated with the indicated concentrations of R406 and then treated without or with 5 mM H₂O₂ for 15 min. Tyrosine-phosphorylated proteins were immunoprecipitated from cell lysates with antibodies against phosphotyrosine. Immune complexes (top) and whole-cell lysates (bottom) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (D) MDA-MB-231-TR (TR) or MDA-MB-231-TRS cells induced to express Syk-EGFP (TRS) were treated without or with 5 mM H₂O₂ for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (E) DG75 B cells (−) or DG75 cells stably expressing shRNA targeted against Syk (+) were treated without or with 5 mM H₂O₂ for 15 min. Nucleolin was immunoprecipitated, and the resulting immune complexes were probed by Western blotting for phosphotyrosine (top) or NCL (bottom). (F) Syk-EGFP (Syk) or Syk-EGFP(K396R) (KD) was immunoprecipitated from the corresponding doxycycline-induced lines of MDA-MB-231 cells using GFP-nanotrap beads. The resulting immune complexes were incubated with buffer containing (+) or lacking (−) ATP. The immune complexes and whole-cell lysates were separated by SDS-PAGE and analyzed by Western blotting with antibodies against NCL. (G) Nucleolin was immunoprecipitated from Tet-responsive MDA-MB-231 cells either uninduced (−) or induced (+) with doxycycline to express Syk-EGFP and either treated with 5 mM H₂O₂ for 3 h or not treated. Immune complexes were examined for the presence of Bcl-x_L mRNA by RT-PCR (top) and nucleolin by Western blotting (middle). The expression of Syk-EGFP was determined by Western blotting of whole-cell lysates with antibodies against Syk (bottom). (H) The relative amount of Bcl-x_L mRNA associated with nucleolin, analyzed as described in the legend to panel G, was quantified. The data represent means ± SEMs from three replicate experiments. The level of mRNA bound to nucleolin in Syk-EGFP-expressing cells not treated with H₂O₂ was set equal to a value of 1.0.