SorLA interacts with the BCR in Ramos B and primary B cells. (A) Representative confocal micrographs of SorLA (green) and anti-IgM surrogate antigen (magenta) following 5-min incubation at 37°C. 100× magnification; inset (blue box) shows an enlarged view of an individual cell. Colocalization appears white. Scale bars = 10 µm. (B) Quantification of A by Manders’ colocalization coefficient. n = 159 cells from five images. Data are shown as box and whisker plots, where the line indicates the median, the box represents the interquartile range, and whiskers denote the minimum and maximum values. (C) Anti-IgM immunoprecipitation in primary B cells. Representative immunoblot of two independent experiments. Cell lysates were incubated with biotinylated anti-IgM F(ab′)₂ or biotinylated anti-CD43, as a negative control not expressed in B cells, and Dynabeads M-280 Streptavidin. Immunoprecipitation eluate was analyzed by western blot (probed with anti-SorLA antibody). n = 2 of two pooled mice per sample. (D) SPR response at different concentrations of human IgM (red) and IgG (blue) to immobilized His-tagged SorLA (captured at 10 µg/ml). Two independent experiments. (E) Representative SPR sensorgram showing blank- and reference cell–subtracted data for concentration-dependent binding of human Ig to immobilized His-tagged SorLA (captured at 10 µg/ml). Increasing analyte concentrations (indicated) were injected over the sensor surface, and binding responses were recorded in response units (RU) over time. The approximate dissociation constants were determined to be K_D ≈ 495–668 nM and 1.84–2.2 µM for IgG and IgM, respectively. (F) Representative SORL1 knockout immunoblot showing total SorLA protein expression in Ramos-Cas9 cell lines expressing one of three different NTg sgRNAs or two sgRNAs targeting SORL1. GAPDH used as protein-loading control. (G) Quantification of SorLA protein depletion per sgRNA-transduced cell line (indicated by symbols) relative to mean SorLA expression in NTg Ramos-Cas9 cell lines per experiment. SorLA depletion quantified by densitometry of SORL1 knockout immunoblots. n = 4–12 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by symbols), quantified from four immunoblots. Data show mean ± SEM. P < 0.0001 calculated by unpaired t test. (H) Representative immunoblot showing total IgM expression in NTg and SORL1^KO Ramos-Cas9 cell lines. GAPDH used as protein-loading control. (I) Densitometry quantification of total IgM expression in NTg and SORL1^KO Ramos-Cas9 cell lines relative to mean total IgM expression in NTg Ramos-Cas9 cell lines per experiment. n = 6–9 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by symbols) quantified from three immunoblots. (J) Surface BCR expression for NTg and SORL1^KO Ramos-Cas9 cells relative to mean NTg surface BCR expression per experiment. n = 10–15 from five independent experiments. (K) Surface BCR expression for WT and Sorl1^−/− cells relative to mean WT surface BCR expression per experiment. n = 16-17 mice from three independent experiments. (L) Surface CD19 expression for NTg and SORL1^KO Ramos-Cas9 cells relative to mean NTg surface CD19 expression per experiment. n = 8–11 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by symbols) from four independent experiments. (M) Surface transferrin receptor expression for NTg and SORL1^KO Ramos-Cas9 cell lines relative to mean NTg surface transferrin receptor expression per experiment. n = 6–8 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by symbols) from three independent experiments. (I–M) Data show mean ± SEM. P < 0.0001 calculated by unpaired t test. Source data are available for this figure: SourceData F2.