Fig 1: Ccr2-KO mice exhibit enhanced BCR proximal signalling. Purified splenic B cells were incubated with AF546-F(ab')2-anti-mouse-Ig (M + G) at 4°C for 30 min and activated at 37°C for 5, 10 and 30 min, confocal microscopy (CFm) was performed. Cells were incubated with biotin-conjugated F(ab')2-anti-mouse-Ig (M + G) and streptavidin, then activated at 37°C for 5, 10 and 30 min, western blotting was performed. (A) Representative CFm images of phosphorylated CD19 (pCD19) and BCR (60× objective, scale bar = 2.5 µm). (B) Colocalisation between pCD19 and BCR. (C) Western blotting of pCD19, pSyk, pCD79a expression in B cells. (D) Representative CFm images of pY and BCR (60× objective, scale bar = 2.5 µm). (E) Colocalisation between pY and BCR. (F) Colocalisation between pBTK and BCR. (G) Western blotting of pY expression in B cells. (H) Western blotting of pBTK and BTK expression in B cells. (I) Colocalisation between pSHIP-1 and BCR. (J) Western blotting of pSHIP-1 and SHIP-1 expression in B cells. (K) Western blotting of pBTK and pSHIP-1 in WT B cells, Ccr2-KO B cells and Ccr2-KO B cells treated with 5µM R406. (L) Representative image of intracellular Ca2+ flux kinetics in WT and Ccr2-KO B cells following stimulation with 10 µg/ml biotin-conjugated F(ab')2 anti-mouse Ig (M + G). All images were representative images from three independent experiments. The number of cells analysed for each parameter in CFm assay was 30–50. Error bars were shown as mean (± SD). *p < .05, **p < .01, ***p < .001, ns: no statistical significance