Panel A shows confocal fluorescence microscopy images of anti-Immunoglobulin M colocalization with Rab5, Rab7, Rab11, and Lamp1 vesicles in NTg and SORL1 knockout cells, including merged and three-dimensional views. Panel B shows a bar graph quantifying anti-Immunoglobulin M colocalization with Rab5 and Rab11 vesicles. Panel C shows a bar graph quantifying anti-Immunoglobulin M colocalization with Rab7 vesicles over time. Panel D shows a bar graph quantifying anti-Immunoglobulin M colocalization with Lamp1 vesicles over time. Panel E shows immunoblot images of Lamp1 and GAPDH expression in wildtype and Sorl1 knockout samples. Panel F shows immunoblot images of Lamp1 and GAPDH expression in NTg and SORL1 knockout samples. Panel G shows a bar graph quantifying total Lamp1 levels relative to NTg samples. Panel H shows immunoblot images of anti-Immunoglobulin M degradation over time in NTg and SORL1 knockout cells. Panel I shows a line graph of B-cell receptor degradation over time in NTg and SORL1 knockout cells. Panel J shows a bar graph quantifying the area under the curve for B-cell receptor degradation. Panel K shows immunoblot images of Cathepsin S and GAPDH expression in NTg and SORL1 knockout samples. Panel L shows a bar graph quantifying pro-Cathepsin S and Cathepsin S levels relative to NTg samples.
SorLA regulates antigen-induced BCR degradation in Ramos-Cas9 cells. (A) Representative confocal micrographs of anti-IgM surrogate antigen (magenta) and distinct vesicle markers (cyan) following 15-min incubation at 37°C. 100× magnification. Images show individual fluorescent channels and an overlay from a single z-stack slice (ImageJ). 3D column shows a 3D projection of the overlay z-stack (Imaris). Colocalization appears white. Scale bars = 10 µm. (B) Quantification of anti-IgM colocalization with organelle markers in NTg and SORL1KO Ramos-Cas9 cell lines following surrogate antigen stimulation for 15 min. Manders’ coefficient. Rab5: n = 20–23 cells, Rab11: n = 25–33 cells. Three independent experiments. (C) Quantification of anti-IgM colocalization with Rab7 in NTg and SORL1KO Ramos-Cas9 cell lines following surrogate antigen stimulation for indicated times. Manders’ coefficient. n = 9–51 cells in three independent experiments. (D) Quantification of anti-IgM colocalization with LAMP1 in NTg and SORL1KO Ramos-Cas9 cell lines following surrogate antigen stimulation for indicated times. Voxel-based Manders’ coefficient. n = 29–68 cells in three independent experiments. (E) Representative immunoblot showing total LAMP1 expression in WT and SORL1KO mouse splenic B cells. GAPDH used as protein-loading control. (F) Representative immunoblot showing total LAMP1 expression in NTg and SORL1KO Ramos-Cas9 cell lines. GAPDH used as protein-loading control. (G) Densitometry quantification of total LAMP1 expression in NTg and SORL1KO Ramos cell lines. n = 4–6 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by different symbols) quantified from two immunoblots. P values shown are calculated by unpaired t test. (H) Representative immunoblot showing BCR degradation in NTg and SORL1KO Ramos-Cas9 cell lines over 12 h following surrogate antigen stimulation. No biotin and no anti-IgM controls are included at 6 h. (I) % BCR degradation calculation using densitometry values from BCR degradation immunoblots (n = 5, three independent experiments) in NTg and SORL1KO Ramos-Cas9 cell lines over 24 h following surrogate antigen stimulation. (J) Quantification of the area under curve shown in C relative to NTg cells per experiment. Distinct sgRNAs are indicated by symbols. (K) Representative immunoblot showing total pro-cathepsin S (37 kDa) and cathepsin S (24 kDa) expression in NTg and SORL1KO Ramos-Cas9 cell lines. GAPDH is used as protein-loading control. (L) Quantified total pro-cathepsin S and cathepsin S expression in NTg and SORL1KO Ramos-Cas9 cell lines. Total pro-cathepsin S and cathepsin expression quantified by densitometry of total two independent pro-cathepsin S and cathepsin immunoblots. n = 4–6. P values shown in B, C, D, and L are calculated by two-way ANOVA. P values shown in G and J are calculated by unpaired t test. Data show mean ± SEM. Source data are available for this figure: SourceData F4.