CIN85 knockdown enhances BCR-induced calcium flux and phosphorylation of Syk, Vav2, and PLCγ2, leading to augmented NF-AT activation and CD69 expression. (A-B) Stable control and CIN85-knockdown BJAB cells were stimulated with 20 μg/mL F(ab′) 2 goat anti–human IgM for the indicated time periods. The cell lysates were subsequently separated on a 10% SDS-PAGE gel and analyzed by Western blotting with anti–phospho-Syk pAb, anti-Syk mAb, anti–phospho-BLNK mAb, anti-BLNK mAb, anti–phospho-PLCγ2 pAb, anti-PLCγ2 pAb, anti–phospho-Akt pAb, anti-Akt pAb, or anti-CIN85 mAb. The resulting values are expressed as fold changes in protein expression compared with unstimulated control cells. The values are the mean ± SD of 3 independent experiments (*P < .05, **P < .01 vs controls). (C) Ca2+ influx in stable control and CIN85-knockdown BJAB cells. Intracellular free calcium levels in Fluo 4/AM-loaded cells were analyzed using flow cytometry after the cells were stimulated with 20 μg/mL F(ab′)2 goat anti–human IgM. The results shown are representative of 4 independent experiments. (D-E) Stable control and CIN85-knockdown BJAB cells were stimulated with 20 μg/mL F(ab′)2 goat anti–human IgM for the indicated time periods. Immunoprecipitates with anti-Vav2 or anti–c-Cbl mAb were separated on a 10% SDS-PAGE gel and analyzed by Western blotting with anti-phosphotyrosine mAb, anti-Vav2 mAb, or anti–c-Cbl mAb. The resulting values are expressed as fold changes in protein expression compared with unstimulated control cells. The values are the mean ± SD of 3 independent experiments (*P < .05, **P < .01 vs controls). (F) Stable control and CIN85-knockdown BJAB cells transfected with the NF-AT luciferase reporter construct were stimulated with graded doses of F(ab′)2 goat anti–human IgM for 8 hours and lysed, and the luciferase activity was assayed using a luminometer. The relative luciferase activity of the medium and BCR-stimulated cells was expressed with respect to that of the PMA/ionomycin stimulation. The results were presented as the mean and SEM of triplicate cultures. One experiment representative of 4 independent experiments is shown (*P < .05 vs controls). (G) Stable control and CIN85-knockdown BJAB cells before and after stimulation with 20 μg/mL F(ab′)2 goat anti–human IgM (3 and 5 hours) were analyzed for surface expression of CD69. One experiment representative of 3 independent experiments is shown (**P < .01 vs controls).
