Figure 2.
CD22 expression as assessed by semi-quantitative real-time polymerase chain reaction (SQRT-PCR), Western blot and monoclonal antibody (mAb) Cy34.1 is not affected by expression of FcγRIIb. To determine CD22 expression at the messenger RNA (mRNA) and protein levels, B cells were purified by magnetic cell sorting and cultured with anti-μ F(ab′)2 or lipopolysaccharide (LPS) for 48 hr. Levels of CD22 mRNA were quantified (relative to GAPDH) using SQRT-PCR, and expressed relative to controls for each stimulation condition (a). CD22 protein was detected by Western blotting and normalized to a β-actin loading control by densitometry (b). Binding of the anti-CD22 mAb Cy34.1, to B220+ splenocytes (i and iii), or B-cell lines (ii), was examined by flow cytometry (c). Results for SQRT-PCR and protein analysis are expressed as mean + SD for three independent experiments. P-values refer to a Student's t-test analysis.