PU.1^Δ/ΔCD19 B-cell response to anti-CD40 and LPS. (A) Syndecan-1 expression in B-cell cultures. Small naive B cells were isolated from spleens of PU.1^+/+CD19 and PU.1^Δ/ΔCD19 mice and cultured with either anti-CD40/IL-4/IL-5 or LPS for 3 days, after which the expression of syndecan-1 was assessed. (B) Expression of immunoglobulin isotypes by anti-CD40/IL-4/IL-5- or LPS-stimulated PU.1^+/+CD19 and PU.1^Δ/ΔCD19 B cells. B cells from PU.1^+/+CD19 and PU.1^Δ/ΔCD19 mice were cultured for 3 days, washed, counted, and a known number of cells reseeded and cultured overnight in fresh media containing no stimuli. The supernatants were then harvested and the level of the indicated immunoglobulin isotypes measured by ELISA. (C) Western and RT-PCR analysis of gene expression in PU.1^+/+CD19 and PU.1^Δ/ΔCD19 mice. (Top) Western hybridization of PU.1 in cells following 3 days of stimulation with anti-CD40/IL-4/IL-5 or LPS; β-actin is shown as a loading control. (Bottom) RT-PCR analysis of gene expression in PU.1^+/+CD19 and PU.1^Δ/ΔCD19 cells after 3 days of stimulation. Hprt is shown as a loading control, and a sample of PU.1^+/+CD19 minus RT was used a negative control. (D) Electrophoretic mobility shift assays. Nuclear extracts were made from PU.1^+/+CD19 and PU.1^Δ/ΔCD19 B cells stimulated with LPS and incubated with a radiolabeled oligonucleotide representing the high-affinity PU.1 binding site from the SV40 promoter. Binding of PU.1 was confirmed by the addition of anti-PU.1 and anti-Pax5 antibodies. FP indicates free probe; BP, bound probe; NS, nonspecific; and SS, supershift.