Phosphorylation and distribution of CD22 induced by anti-CD20/22 bsAbs. D1-7 or Ramos cells were treated for 15 minutes with 100 nM of hA20IgG-(RFB4scFv)2, hA20IgG-(LL2scFv)2, epratuzumab or hA20 (veltuzumab). Cells were lysed in buffer containing 0.5% CHAPS and centrifuged to clear the supernatant. CD22 in the supernatants was immunoprecipitated by epratuzumab. The pellet (ie, insoluble cell debris) was solubilized in SDS-PAGE sample buffer. (A) Immunoprecipitated CD22 from D1-7 cells was analyzed by Western blotting with HRP-4G10 to reveal CD22 phosphorylation. The position of phosphorylated CD22 is indicated on the right. (B) Immunoprecipitated CD22 (marked as soluble on left) and insoluble pellet proteins (marked as pellet on left) from D1-7 (top 2 panels) and Ramos (bottom 2 panels) cells were analyzed by Western blotting with a rabbit anti-CD22 Ab to reveal total CD22. The positions of CD22 are indicated on the right. Lanes labeled as α-CD20/22-R and α-CD20/22-L are hA20IgG-(RFB4scFv)2 and hA20IgG-(LL2scFv)2 treated samples, respectively.
