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. 2022 Feb 9;8(6):eabm1759. doi: 10.1126/sciadv.abm1759

Fig. 8. Effect of BCR VDGs on antigen internalization and BCR downmodulation.

Fig. 8.

(A) Ramos B cells were incubated at 4°C, stimulated with antigen or PBS, and incubated at 4° or 37°C to allow BCR downmodulation. The remaining surface BCRs were stained (Fab anti-human IgG-AF647). (B) GFP and mIgG expression of MDL-AID KO, 3F3 WT, and NG cells after PBS or CCP2-strep. treatment (4°C or 15 min at 37°C). (C) Histograms (mIgG) of CCP2-strep.–stimulated 3F3 WT or NG Ramos B cells (4°C or 5, 15, and 30 min at 37°C). (D) Histogram (mIgG) overlay of 3F3 WT and NG Ramos B cells after PBS or CCP2-strep. treatment (4°C or 15 min at 37°C). (E) BCR downmodulation of 3F3 WT and NG (F) 7E4WT and NG after PBS, CCP2-strep., or CArgP2-strep. stimulation (4°C or 5, 15, and 30 min at 37°C). Paired two-tailed t test. N = 3 to 4. 3F3: *(5 min)P = 0.0146 and **P = 0.0054; 7E4: **P = 0.0054. (G) Spinning disk confocal microscopy of GFP+ 3F3 WT and NG cells after CCP2-strep.-AF568 stimulation (incubation at 4°C or 5 and 15 min at 37°C), 2% PFA fixation, and a-IgG-AF647 surface staining. (H) 3F3 WT and NG BCR expression after CCP2-strep. stimulation (4°C or 5 and 15 min at 37°C). N = 114, 213, 474, 224, 495, and 595 cell slices, respectively. Ordinary one-way ANOVA, ****P < 0.0001. MSI, mean signal intensity. (I) CCP2-strep. binding of 3F3 WT and NG at 4°C. (J) CCP2-strep. internalization of 3F3/7E4 WT and NG BCRs at 4°C or after 5 and 15 min of incubation at 37°C. N(3F3) = 114, 213, 474, 224, 495, and 595 cell slices, respectively. N(7E4) = 619, 459, 645, 433, 738, and 302 cell slices, respectively. Ordinary one-way ANOVA, ****P < 0.0001.