LIME is required for BCR-dependent Akt activation. (A) A20 cells were transfected with plasmids expressing siRNAs directed against murine LAB, LIME, or LAT. After 48 hours, transfected cells were activated with 20 μg/mL F(ab′)2 goat anti-mouse IgG for 3 or 10 minutes at 37°C. Lysates were analyzed by Western blotting with anti-phospho-Akt, anti-Akt, or anti-LIME Abs. The level of Lab mRNA was assessed by RT-PCR. (B) p85 associates with LIME. A20 cells were transfected with the plasmids expressing LIME-flag or LAB-flag and stimulated with 20 μg/mL F(ab′)2 goat anti-mouse IgG for 0 or 1 minutes at 37°C. The lysates were immunoprecipitated using an antiflag Ab, and the precipitates were analyzed by Western blotting using an anti-p85 or antiflag Ab. (C) p85 was coexpressed with LIME-flag or LAB-flag in 293T cells in combination with Lyn or Syk. Lysates were subjected to immunoprecipitation using an antiflag Ab, and the precipitates were analyzed by Western blotting using an anti-p85 or antiflag Ab. (D) Mapping of the motifs responsible for association with p85. Flag-tagged tyrosine mutants of LIME were coexpressed with p85 and Lyn in 293T cells. Lysates were subjected to immunoprecipitation using an antiflag Ab, and the precipitates were analyzed by Western blotting using an anti-p85 or antiflag Ab.
