CLL cells from Eμ-TCL1 mice express a constitutively active BCR, which may be a result of altered glycosylation of the BCR. (A) Wild-type B cells and CLL cells isolated from 8-month-old wild-type and Eμ-TCL1 mice were cultured in the presence of LPS for 3 days. Their BCR is subsequently activated by F(ab′)2 fragments of the goat anti–mouse IgM Ab for 2 minutes. Cells were immediately lysed for analysis by immunoblots using Abs against indicated molecules. Data shown in each immunoblot are representative of 3 independent experiments. For each experiment, CD5⁻/B220⁺ wild-type B cells and CD5⁺/B220⁺ Eμ-TCL1 CLL cells were purified and pooled from at least 2 mouse spleens. (B) Wild-type B cells and CLL cells isolated from 8-month-old wild-type and Eμ-TCL1 mice were stimulated with LPS for indicated days and lysed for analysis by immunoblots using Abs to immunoglobulin μ heavy chain, Igα, Igβ, and actin. Data shown in each immunoblot are representative of 3 independent experiments. For each experiment, CD5-/B220⁺ wild-type B cells and CD5⁺/B220⁺ Eμ-TCL1 CLL cells were purified and pooled from at least 2 mouse spleens. (C) Wild-type B cells and Eμ-TCL1 CLL cells purified from 8-month-old mice were radiolabeled for 15 minutes, chased for the indicated time, and lysed. Intracellular IgM was immunoprecipitated from the lysates using an anti-μ Ab, and analyzed on an SDS-PAGE gel. Data are representative of 3 independent experiments. For each experiment, wild-type B cells and Eμ-TCL1 CLL cells were purified and pooled from 2 mouse spleens. (D) Similarly, extracellular sIgM was immunoprecipitated from culture media using an anti-μ or an anti-κ Ab and analyzed by SDS-PAGE. Data are representative of 3 independent experiments. (E) Using similar lysates as those in panel C, we performed immunoprecipitations using an anti-Igβ Ab to retrieve the Igα/Igβ heterodimers. Immunoprecipitated proteins were eluted from the beads and treated with endo-H or PNGase F before analysis by SDS-PAGE. CHO, CHO*, NAG represent high mannose-type glycans, complex-type glycans, and N-acetylglucosamines, respectively. Data are representative of 3 independent experiments. (F) Similar lysates (as panel C) were immunoprecipitated using an Ab against the class I MHC heavy chain (HC), and immunoprecipitates were analyzed by SDS-PAGE. Data are representative of 3 independent experiments.