Panel A shows immunoblots for phosphorylated Syk, total Syk, and GAPDH in NTg and SORL1KO Ramos-Cas9 B cell lines with and without anti-IgM stimulation. Panel B displays flow cytometry histograms for phospho-Syk in different conditions. Panel C is a bar graph showing p-Syk fold change in NTg and SORL1KO cells. Panel D presents immunoblots for phosphorylated Akt1, total Akt1, phosphorylated Erk1/2, total Erk1/2, and GAPDH. Panel E is a bar graph of p-Akt1 Ser473 fold change, while Panel F shows p-Akt1 Thr308 fold change. Panel G is a line graph depicting Akt1 phosphorylation at Ser473 over time. Panel H is a line graph of Akt1 phosphorylation at Thr308 over time. Panel I is a bar graph of p-Erk1/2 fold change. Panel J shows immunoblots for Rab11 and GAPDH. Panel K displays immunoblots for Rab7 and GAPDH. Panel L shows flow cytometry histograms for different conditions. Panel M is a bar graph of recycled IgM in NTg and SORL1KO cells.
SorLA does not play a significant role in BCR signalling or recycling. (A) Representative immunoblot showing total and phosphorylated Syk (p-Syk) expression in NTg and SORL1KO Ramos-Cas9 B cell lines following 5-min incubation at 37°C with or without 5 μg/ml F(ab′)2 anti-IgM stimulation. GAPDH used as protein-loading control. (B) Flow cytometry population histograms for phospho-Syk showing unstained (blue), secondary antibody only (red), unstimulated (orange), and stimulated (green). (C) p-Syk phospho-flow. p-Syk fold change in NTg and SORL1KO Ramos-Cas9 cells at 5 min, relative to 0-min time point. n = 10–15 independent Ramos-Cas9 transductions with three NTg sgRNAs and two SORL1-targeting sgRNAs (indicated by different symbols) from six independent experiments. (D) Representative immunoblot showing total and phosphorylated Akt1 (Ser473 and Thr308) and Erk1/2 expression in NTg and SORL1KO Ramos-Cas9 B cell lines following 15-min incubation at 37°C with or without 5 μg/ml F(ab′)2 anti-IgM stimulation. GAPDH used as protein-loading control. (E) p-Akt1 Ser473 fold change in NTg and SORL1KO Ramos-Cas9 B cell lines, relative to unstimulated. p-Akt1 Ser473 fold change quantified by densitometry of a p-Akt1 Ser473 immunoblot. n = 5–8 in two independent immunoblots. P value shown are calculated by Mann–Whitney U test. (F) p-Akt1 Thr308 fold change in NTg and SORL1KO Ramos-Cas9 cell lines, relative to unstimulated. p-Akt1 Thr308 fold change quantified by densitometry of a p-Akt1 Thr308 immunoblot. n = 2–3. (G) Akt1 phosphorylation at Ser473 over 30 min per phospho-flow time course experiment, n = 2–3 per experiment, two independent experiments. Background MFI subtraction using secondary only control. (H) Akt1 phosphorylation at Thr308 over 30 min per phospho-flow time course experiment, n = 2–3 per experiment, two independent experiments. Background MFI subtraction using secondary only control. (I) p-Erk1/2 fold change in NTg and SORL1KO Ramos-Cas9 B cell lines, relative to unstimulated. p-Erk1/2 fold change quantified by densitometry of a p-Erk1/2 immunoblot. n = 4. (J) Immunoblot showing total Rab11 expression in NTg and SORL1KO Ramos-Cas9 cell lines. GAPDH used as protein-loading control. (K) Immunoblot showing total Rab7 expression in NTg and SORL1KO Ramos-Cas9 cell lines. GAPDH used as protein-loading control. (L) Flow cytometry population histograms for unstained (orange), ice (blue), 37°C (red), and maximum streptavidin (green). (M) Recycled IgM in NTg and SORL1KO Ramos-Cas9 cell lines relative to mean NTg surface recycled IgM per experiment. P value shown are calculated by unpaired t test, n = 4–5 from two independent experiments. P values shown in C, F, and I are calculated by unpaired t test. Data in C, F–I, and M show mean ± SEM. Source data are available for this figure: SourceData FS1.