Research Article

Internalization of B Cell Receptors in Human EU12 μHC+ Immature B Cells Specifically Alters Downstream Signaling Events

Figure 3

Internalization of BCR changes downstream signaling events upon restimulation. (a) Schematic representation of different treatment for EU12 μHC+, Daudi, or Ramos cells with goat anti-IgM antibody F(ab′)2 fragments. (b) EU12 μHC+, Daudi, and Ramos cells were directly stimulated with goat F(ab′)2 anti-IgM antibodies (10 μg/mL or 20 μg/mL) or pretreated with goat anti-IgM antibody F(ab′)2 fragments (2 μg/mL) for 30 min to internalize BCRs, washed to remove excessive anti-IgM antibodies, recovered for 2 h or 4 h, and then restimulated with goat anti-IgM antibody F(ab′)2 fragments (10 μg/mL). BCR-mediated downstream signaling events were monitored by Western blot analyses using 4G10 (p-Tyr) antibodies or antibodies specific to phosphorylated CD19 (p-CD19), phosphorylated BLNK (p-BLNK), phosphorylated ERK (p-ERK), phosphorylated AKT (p-AKT), or phosphorylated FOXO1/3a (p-FOXO1/3a). Membranes were stripped and reprobed with antibodies against CD19, Lyn, SYK, BLNK, ERK, AKT, FOXO1, and β-ACTIN as controls.