Expression of SAP in B cells. (A) Expression of the Sap transcript. RT-PCR products with primers 91U and 457L²³  from 1μg total RNA extracted from the following cells: thymocytes (T), splenocytes (S), sorted CD3⁺ cells (CD3), sorted CD19⁺ cells (B1), negatively selected B cells, unstimulated (B2), and stimulated by LPS+IL-4 for 3 days (D3) and 6 days (D6), respectively. Mouse fibroblast cell line NIH 3T12 (F) and mouse neuroblastoma cell line Neuro 2A (N) were used as negative controls for Sap expression. The transcript of the mouse β-actin gene was used as the amplification control, and the transcript of mouse CD3 as the purity control of the enriched B cells. (B) Expression of the SAP protein. Top panel: SAP protein detected from thymocytes (T), splenocytes (S), T cells selected by Dynal beads (T cell), negatively selected B cells, unstimulated (B), and stimulated with LPS+IL-4 for 6 hours (6 h), 18 hours (18 h), 3 days (D3), and 6 days (D6), respectively; bottom panel: SAP protein detected from negatively selected wild-type B cells (B cells SAP⁺) and from negatively selected Sap-deficient B cells (B cells SAP^-) mixed with wild-type T cells mimicking various percentages of T-cell contamination. The same amount of cell extract (0.5 mg protein) was immunoprecipited by anti-SAP antibody and the Western blot was hybridized with the same anti-SAP antibody.