(A) Genetic approach used to delete ITSN2. A LacZ cassette was inserted in the Itsn2 locus to disrupt protein expression. A neomycin resistance cassette flanked by two loxP sites was used as a selection marker, and subsequently excised by Cre-mediated recombination (KOMP allele tm1.1). (B) Naïve B cells were purified from the spleens of WT and Itsn2-/- mice and protein expression of ITSN2 (upper immunoblot, two isoforms as illustrated on the right) and clathrin heavy chain (lower immunoblot) were detected by Western blot. (C) Bone marrow from WT and Itsn2-/- littermates was analysed by flow cytometry using the gating strategy shown on the left. B cell progenitors (B220+) were divided into immature (CD43+) and mature (CD43-) cells on the basis of CD43 expression. Data quantified in the panels on the right show the percentage of live cells in the indicated gates. (D) Thymus from WT and Itsn2-/- littermates were analysed by flow cytometry. Gating strategy is shown on the left. T cell progenitors (CD3+) were divided into double negative (CD4- CD8-), double positive (CD4+ CD8+), CD4 single positive (CD4+ CD8-) and CD4 single positive (CD4- CD8+) cells. Data quantified in the panels on the right show the percentage of CD3+ cells in the indicated gates. (E–H) Spleens from WT and Itsn2-/- were analysed by flow cytometry. (E) Identification and quantification of B cells (B220+ TCRβ−) and T cells (B220- TCRβ+). (F) T cells were subdivided into CD4 (CD4+ CD8-) and CD8 (CD4- CD8+) T cells and quantified. (G–H) B cells were divided on the basis of CD21, CD23 and CD24 expression into T0/T1 cells (CD21-CD24hi, G), follicular B cells (CD21loCD24lo, G), T2 cells (CD21hi CD24hi CD23-, H), MZB cells (CD21hi CD24hi CD23+, H) and quantified (right panels). For all flow cytometry experiments (C–G), data are from 1 out of 3 representative experiment with 4 or more animals in each group, and each dot represents an individual mouse. Student’s t-test, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001.