Figure 2.
BCR signaling is attenuated in INPP5B-depleted cells. (A) Protein extracts from BCR-stimulated INPP5BDegron/Degron cells were blotted for ERK and Akt phosphorylation at the indicated time points. Quantification of three such blots by densitometry is shown on the right. Error bars represent SEM. Data were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. (B) GTP-bound Ras was detected using GST-Raf RBD pulldown followed by blotting against Ras. Quantification of three such experiments by densitometry is shown on the right. GTP levels of Ras are expressed relative to total Ras. Error bars represent SEM, and data were analyzed as in A. (C) Representative blot from INPP5BDegron/Degron cells that were stimulated on coverslips presenting surrogate antigens for 5 min. Quantification of three such blots is shown on the right. Data were analyzed as in A. (D) PI(3,4,5)P3 levels in INPP5B-depleted cells. INPP5BDegron/Degron cells were treated as in A; however, they were stimulated for the times indicated before analysis by MS. PI(3,4,5)P3 levels are expressed relative to PI (see Materials and methods), and data were combined for C36:2, C38:3, and C38:4 species. Error bars represent SD, and data were analyzed as in A. (E) Representative pCD19 and pSyk blots from BCR-stimulated INPP5BDegron/Degron cells at the indicated time points. Quantification of three such blots is shown on the right. Data were analyzed as in A. (F) DAG levels are expressed relative to internal standard (see Materials and methods), and data were combined for C38:4 and C34:1 species. Error bars represent SEM, and data were analyzed as in A. (G) Representative single-cell intracellular Ca+2 responses to anti-IgM (added at arrow) in vehicle- and auxin- treated INPP5BDegron/Degron cells. (H) Left: Each bar represents peak fluorescence (Fura-2) ratio of anti-IgM response expressed as mean ± SEM (n ≥ 20 cells from three independent experiments on three different days). Middle: Percentage of cells that displayed spontaneous (Ca2+)i oscillations in total anti-IgM stimulating responding cells. Right: Spontaneous (Ca2+)i oscillation frequency observed in vehicle- and auxin-treated INPP5BDegron/Degron cells. Source data are available for this figure: SourceData F2.

BCR signaling is attenuated in INPP5B-depleted cells. (A) Protein extracts from BCR-stimulated INPP5BDegron/Degron cells were blotted for ERK and Akt phosphorylation at the indicated time points. Quantification of three such blots by densitometry is shown on the right. Error bars represent SEM. Data were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. (B) GTP-bound Ras was detected using GST-Raf RBD pulldown followed by blotting against Ras. Quantification of three such experiments by densitometry is shown on the right. GTP levels of Ras are expressed relative to total Ras. Error bars represent SEM, and data were analyzed as in A. (C) Representative blot from INPP5BDegron/Degron cells that were stimulated on coverslips presenting surrogate antigens for 5 min. Quantification of three such blots is shown on the right. Data were analyzed as in A. (D) PI(3,4,5)P3 levels in INPP5B-depleted cells. INPP5BDegron/Degron cells were treated as in A; however, they were stimulated for the times indicated before analysis by MS. PI(3,4,5)P3 levels are expressed relative to PI (see Materials and methods), and data were combined for C36:2, C38:3, and C38:4 species. Error bars represent SD, and data were analyzed as in A. (E) Representative pCD19 and pSyk blots from BCR-stimulated INPP5BDegron/Degron cells at the indicated time points. Quantification of three such blots is shown on the right. Data were analyzed as in A. (F) DAG levels are expressed relative to internal standard (see Materials and methods), and data were combined for C38:4 and C34:1 species. Error bars represent SEM, and data were analyzed as in A. (G) Representative single-cell intracellular Ca+2 responses to anti-IgM (added at arrow) in vehicle- and auxin- treated INPP5BDegron/Degron cells. (H) Left: Each bar represents peak fluorescence (Fura-2) ratio of anti-IgM response expressed as mean ± SEM (n ≥ 20 cells from three independent experiments on three different days). Middle: Percentage of cells that displayed spontaneous (Ca2+)i oscillations in total anti-IgM stimulating responding cells. Right: Spontaneous (Ca2+)i oscillation frequency observed in vehicle- and auxin-treated INPP5BDegron/Degron cells. Source data are available for this figure: SourceData F2.

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