CD33 expression is also detected by Western blot and RT-PCR analysis. (A) A 67-kDa protein revealed by antibody WM53 and corresponding to CD33 was detected by immunoblot analysis of tumoral pDCs from patients no. 2, 3, and 4 (P2, P3, P4, respectively). TP1 and TP2 (2 AML) were used as positive controls, and TN1 (human fibrosarcoma cell line HT1080) was used as a negative control. The loading and transfer of equivalent protein levels was confirmed using β-actin mAb staining. Densitometric analysis of CD33 and β-actin signal and determination of CD33 signal/β-actin signal ratio demonstrated that cells from patients no. 2 and 3 expressed similar levels of CD33 (CD33/β-actin ratio 2.4 and 3.4, respectively) as TP1 and TP2 (CD33/β-actin ratio 2 and 2.2, respectively). The CD33/β-actin ratio for cells from patient no. 4 was lower (1.0 compared to 0 for TN1). (B) Expression of CD33 mRNA transcripts (305 bp) was detected in the tumoral pDCs from the 4 patients (P1, P2, P3, and P4).As in panelA, positive controls were AML cells (TP1 and TP2) and negative control was the HT1080 line. Two other controls were performed (no addition of DNA, noted as H2O; a control for the absence of gDNA amplification, noted as gDNA). Amplification of a 148-bp c-raf fragment is shown as a control. No contamination with gDNA is attested by the absence of signal at 258 bp.