Reduced IgE/antigen induced FcϵRI-SFK assoctiation and FcϵRI interchain interactions in PigA-deficient BMMCs. (A) Littermate control (WT) or PigA-deficient (KO) BMMCs were sensitized with IgE anti-DNP for 3 hours at 37°C, and either mixed with DNP-BSA on ice (0 minutes), or further incubated with prewarmed DNP-BSA at 37°C for 1 or 3 minutes before transfer to lysis buffer on ice. Top panels: FcϵRI receptor complexes were immunoprecipitated from lysates by anti-IgE (IP: IgE), and immunoblotted with antibodies against FcϵRI α-chain (α), γ-chain (γ), or β-chain (β); or against Lyn (Lyn) or activated SFK (pSrc). In the pSrc blot, the two bottom bands presumably represent Lyn, the upper band Fyn, based on their molecular weight values (53/56 kDa and 59 kDa, respectively). Bottom panels: whole cell lysates (WCL) were immunoblotted with antibodies against FcϵRI α-chain (α), γ-chain (γ), or β-chain (β); data representative of at least two experiments are shown. (B) Quantitative scanning of multiple coimmunoprecipitation experiments specified in panel A, confirming significantly reduced coprecipitation of the γ-chain and β-chain with the receptor complex on stimulation. Shown are signal intensities of anti-IgE immunoprecipitates which were immunoblotted with antibodies against FcϵRI γ-chain (middle panel; n = 4) or β-chain (bottom panel; n = 3); or against α-chain as control (top panel; n = 3). Data are expressed as mean percentages of pixel intensities ± SEM, compared with nonstimulated cells (T = 0). Asterisks indicate significant differences (P < .05) compared with WT cells. (C) IgE binding to control (filled lines) or PigA-deficient (bold lines) BMMCs after IgE sensitization followed by 1 or 3 minutes antigen stimulation.