Defective p66Shc expression in CLL B cells. Quantification by real-time RT-PCR of the levels of p66Shc mRNA in monocyte-depleted peripheral blood mononuclear cells (B cells ≥ 85%, as assessed by flow cytometry) purified by density gradient centrifugation from 64 patients with CLL, of which 32 with mutated IGHV (M-CLL; A) and 32 with unmutated IGHV (U-CLL; B). Transcript levels were normalized to the expression level of GAPDH. For each patient, Sybr green runs were performed on duplicate samples of cDNAs from 2 independent reverse transcription reactions, each carried out on 400 ng of total RNA. RNA from the same healthy donor (C1) was included in duplicate in each run as reference to normalize the data obtained in the individual experiments. The ΔΔC_T method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on healthy donors. (C) Histograms summarizing the real-time RT-PCR analysis of p66Shc mRNA levels in B cells from the 21 healthy donors and 64 patients with CLL shown in panels A and B, grouped in the M-CLL and U-CLL subsets (***P ≤ .001). Error bars indicate SD. (D) Immunoblot analysis of p66Shc expression in lysates of monocyte-depleted peripheral blood mononuclear cells purified by density gradient centrifugation from 4 representative patients with CLL and 1 representative healthy donor. Stripped filters were reprobed with anti-actin antibodies as loading control. The histogram on the right of the panel shows the quantification by laser densitometry of p66Shc immunoreactive band in lysates of PBLs from 19 patients with CLL compared with lysates of peripheral blood B cells purified from 4 healthy donors by immunomagnetic sorting, of which at least 1 was included in each gel as reference (***P ≤ .001).