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[Preprint]. 2023 Mar 15:2023.03.14.532631. [Version 1] doi: 10.1101/2023.03.14.532631

PMC10055065.1; 2023 Mar 15
PMC10055065.2; 2023 Sep 1

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Figure 9. — Primary B-cells from flox control and cNKO mice were incubated with AF546-Fab’-PLB at 37°C, fixed at 3 or 7 min, stained for total SHIP-1 and phosphorylated SHIP-1 (pSHIP-1 Tyr1020), and imaged by TIRF. (A-C) Ratios of SHIP-1 MFI relative to AF546-Fab’ MFI were plotted against AF546-Fab’ peak IF in individual AF546-Fab’ clusters in the contact zone of Flox control (A) and cNKO B-cells (B). An overlay of flox control and cNKO plots is also shown (C). AF546-Fab’ clusters were identified as described in Figure 6 and Figure 6-figure supplement 2 from an equal number of cells after 3 and 7 min stimulation. Data points represent individual AF546-Fab’ clusters with an equal number of clusters from each time point. The black diamond symbols represent the average ratios of SHIP-1 MFI to Fab’ MFI in individual AF546-Fab’ clusters within indicated Fab’ peak FI ranges. The brown square symbols represent the fraction of the AF546-Fab’ clusters out of the total at indicated Fab’ peak FI ranges. (D-F) Ratios of pSHIP-1 MFI relative to AF546-Fab’ MFI were plotted against AF546-Fab’ peak IF in individual AF546-Fab’ clusters in the contact zone of flox control (D) and cNKO B-cells (E) and their overlay (F). (G-I) MFI ratios of pSHIP-1 relative to AF546-Fab’ were plotted against AF546-Fab’ peak IF in individual pSHIP-1 puncta in the contact zone of Flox control (G) and cNKO B-cells (H) and their overlay (I). The black diamond symbols represent the average ratios of pSHIP-1 MFI to Fab’ MFI in individual AF546-Fab’ clusters (D-F) or individual pSHIP-1 puncta (G-I) at indicated Fab’ peak FI ranges. The brown square symbols represent the fraction of the total clusters at indicated Fab’ peak FI ranges. pSHIP-1 puncta were identified using the criteria: FI ≥1.5 fold of the background outside the B-cell contact zone and diameter ≥250 nm. Data points represent individual AF546-Fab’ clusters (D-F) and pSHIP-1 puncta (G and H), collected from an equal number of cells at 3- and 7-min. Data were generated from 3 independent experiments with ~23 cells per condition per experiment. * p <0.05, ** p <0.01, *** p<0.001, by non-parametric student’s t-test, between AF546-Fab’ cluster group with different Fab’ peak FI ranges. The p-values in C, F, and I were corrected using the Benjamini-Hochberg/Yekutieli method for false discovery rate control.