Extended Data Fig. 2: B-cell developmental defect due to spontaneous protein degradation in vivo by the Rosa26Tir1 allele, but not by the Rosa26Tir1-F74G allele.
a, Impaired B-lymphopoiesis in Pax5Aid/Aid Rosa26Tir1/Tir1 mice in the absence of auxin (IAA). B-cell development in the bone marrow of 3-4-week-old Pax5Aid/Aid Rosa26Tir1/Tir1, Pax5Aid/Aid Rosa26Tir1-F74G/Tir1-F74G, and control Pax5+/+ mice was investigated by flow-cytometric analysis. The percentage of B-cell types is indicated next to the gates. Pax5 protein expression in pro-B cells was determined by intracellular anti-Pax5 staining. One of two independent experiments is shown. b, No effect of the Rosa26Tir1-F74G allele in the absence of 5-Ph-IAA treatment on B-cell development in Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, Ikzf1Aid/Aid, Ikzf3Aid/Aid, and Ikzf1,3Aid/Aid mice expressing the Rosa26Tir1-F74G allele. Frequencies of total B, pro-B, pre-B, and immature B cells were analyzed by flow cytometry and presented as mean values with SEM (n = 32,24,13,22,20,7,4). Statistical data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test: *P < 0.05, **P < 0.01, ****P < 0.0001. Each dot corresponds to one mouse. c, Time course analysis of Ikaros degradation for up to 25 h upon 5-Ph-IAA injection into Ikzf1Aid/+ Rosa26Tir1-F74G/+ mice. Aid-V5-tagged Ikaros protein levels were determined by flow-cytometric analysis of intracellular anti-V5 straining. The kinetics of Ikaros degradation is shown as geometric mean fluorescence intensity (gMFI) of the anti-V5 staining in pro-B, small pre-B and immature B cells relative to the gMFI of the untreated control (left; n = 2). One of two independent 25-hour experiments is shown. The histogram displays the Ikaros protein levels in the three B-cell types at the indicated time points after 5-Ph-IAA injection (right). d, Flow-cytometric sorting of pro-B, small pre-B, and immature B cells from the bone marrow of 4-week-old Ebf1Aid/Aid Rosa26Tir1-F74G/Tir1-F74G mice (upper row). The different gates used for flow-cytometric sorting are indicated together the percentage of cells in the respective gates. The purity of the sorted pro-B cell population was determined by reanalysis (lower row).
