Figure 1.
Impaired BCR clustering and accelerated BCR endocytosis in INPP5B-depleted cells. (A) INPP5BDegron/Degron cells were treated with auxin as indicated to induce INPP5B degradation, and the remaining INPP5B was immunoprecipitated against its FLAG tag and detected by Western blot using anti poly-His. Quantification of three such blots by densitometry is shown on the right. Data were analyzed by one-way ANOVA, and P values were calculated using Dunnett’s multiple comparisons test. Error bars represent SD. (B) Representative confocal images at the indicated time points from INPP5BDegron/Degron cells stimulated with Texas Red–conjugated anti-IgM antibody. Scale bar, 10 μm. See Video 1 for the complete time-lapse confocal images. Quantification of cells showing BCR capping at ∼3 min is shown on the right, where a minimum of 50 cells per experiment were examined, and data are expressed as a percentage of cells showing BCR capping events. Data are from four independent experiments. Error bars represent SD, and P value was calculated using Welch’s t test. (C) Representative histograms at the indicated time points showing the clearance of BCR from the cell surface in INPP5BDegron/Degron cells in response to anti-IgM stimulation. (D) Quantification of BCR endocytosis by flow cytometry in INPP5BDegron/Degron cells. Data are from five independent experiments. Error bars represent SD, and P values were calculated using Sidak multiple comparisons test. (E) Representative TIRF microscopy images at the indicated time points of BCR clustering in INPP5BDegron/Degron cells that were stimulated on antibody-coated glass. Cells were prelabeled with Fab fragment anti-IgM. See Video 2 for the complete time-lapse TIRF microscopy images. (F) Quantification of the number of BCR microclusters per cell is shown in F. Data (n = 15 cells) were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. (G) Representative TIRF microscopy images from BCR-labeled NPP5BDegron/Degron cells that were settled on coverslips presenting surrogate antigens for 10 min. F-actin was stained with Phalloidin, and BCR microclusters are denoted by black circles. Scale bar, 10 μm. (H–K) Statistical analyses of the number of BCR microclusters per cell, cell spreading area, TFI, and MFI of BCR staining from vehicle-treated and auxin-treated INPP5BDegron/Degron cells in H–K, respectively. The data represent means ± SD of 35 cells. P values were calculated using Welch’s t test. Source data are available for this figure: SourceData F1.

Impaired BCR clustering and accelerated BCR endocytosis in INPP5B-depleted cells. (A) INPP5BDegron/Degron cells were treated with auxin as indicated to induce INPP5B degradation, and the remaining INPP5B was immunoprecipitated against its FLAG tag and detected by Western blot using anti poly-His. Quantification of three such blots by densitometry is shown on the right. Data were analyzed by one-way ANOVA, and P values were calculated using Dunnett’s multiple comparisons test. Error bars represent SD. (B) Representative confocal images at the indicated time points from INPP5BDegron/Degron cells stimulated with Texas Red–conjugated anti-IgM antibody. Scale bar, 10 μm. See Video 1 for the complete time-lapse confocal images. Quantification of cells showing BCR capping at ∼3 min is shown on the right, where a minimum of 50 cells per experiment were examined, and data are expressed as a percentage of cells showing BCR capping events. Data are from four independent experiments. Error bars represent SD, and P value was calculated using Welch’s t test. (C) Representative histograms at the indicated time points showing the clearance of BCR from the cell surface in INPP5BDegron/Degron cells in response to anti-IgM stimulation. (D) Quantification of BCR endocytosis by flow cytometry in INPP5BDegron/Degron cells. Data are from five independent experiments. Error bars represent SD, and P values were calculated using Sidak multiple comparisons test. (E) Representative TIRF microscopy images at the indicated time points of BCR clustering in INPP5BDegron/Degron cells that were stimulated on antibody-coated glass. Cells were prelabeled with Fab fragment anti-IgM. See Video 2 for the complete time-lapse TIRF microscopy images. (F) Quantification of the number of BCR microclusters per cell is shown in F. Data (n = 15 cells) were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. (G) Representative TIRF microscopy images from BCR-labeled NPP5BDegron/Degron cells that were settled on coverslips presenting surrogate antigens for 10 min. F-actin was stained with Phalloidin, and BCR microclusters are denoted by black circles. Scale bar, 10 μm. (H–K) Statistical analyses of the number of BCR microclusters per cell, cell spreading area, TFI, and MFI of BCR staining from vehicle-treated and auxin-treated INPP5BDegron/Degron cells in H–K, respectively. The data represent means ± SD of 35 cells. P values were calculated using Welch’s t test. Source data are available for this figure: SourceData F1.

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