Fig. 4: CEACAM1 co-localizes to lipid rafts and promotes F-actin reorganization during BCR activation.

a Diagram of sucrose-density gradient fractionation of lipid rafts. b Left panels, Immunoblots of fractions indicated in (a) from control or CEACAM1-knockout JEKO-1 cells stimulated with control or anti-IgM antibody (1 μg/ml) for 2 min. Right panels, immunoblot signal quantification of fraction I (red dashed-line box) after normalization to Flotillin-1 and unstimulated controls. Shown are the means of fold changes from three independent experiments. Error bars, SD. **P < 0.01, *P < 0.05 by a two-sided, paired t-test. ns not significant. c Left panels, Control (gNTC) or CEACAM1 knockout (gCC1) JEKO-1 cells were stimulated with 2 μg/ml anti-IgM antibody for 2 min. Shown are representative cells from confocal immunofluorescence images in Supplementary Fig.8 of control and IgM-stimulated cells co-stained with anti-FLNA (green) and anti-LYN (red) antibodies, followed by nuclear staining with DAPI (blue). Scale bar, 2 μm. Right panels, quantified fluorescent signals for each cell from the samples described. Shown are the sums of intensities in signal-positive areas per cell in arbitrary units from three independent experiments. Horizontal red bars indicate the mean. Approximately 200 cells from each sample were analyzed. ****P < 0.0001 by a two-sided unpaired t-test. ns not significant. d CEACAM1 is required for lipid-raft assembly. Top panels, Control, CEACAM1 knockout (CC1 KO), or CC1 KO + CEACAM1-4L (4 L) JEKO-1 cells were stimulated with 2 μg/ml anti-IgM antibody for 5 min. Shown are representative confocal immunofluorescence images of control and IgM-stimulated cells stained with anti-CEACAM1 antibody (green), GM1 via cholera toxin B (red), p-SRC^Y416 antibody (green), or F-actin via Actin-Stain 555 Phalloidin (red) followed by nuclear staining with DAPI (blue). Scale bar, 10 μm. Bottom panels, quantified fluorescent signals from the samples described in the top panels. Shown are the sums of intensities in signal-positive areas per cell in arbitrary units from three independent experiments. Horizontal red bars indicate the mean. Approximately 200 cells from each sample were analyzed. ****P < 0.0001 by a two-sided unpaired t-test. Source data are provided as a Source Data file.