American Society of Hematology

Figure 1

$p66Shc acts as a negative regulator of B-cell survival. (A) Detection of p66Shc-specific mRNA in peripheral blood B cells purified from healthy donors by semiquantitative RT-PCR. The identity of the band was confirmed by automated sequencing. (B) Immunoblot analysis of p66Shc expression in lysates of peripheral blood B cells purified from 4 representative healthy donors. Lysates of Ramos B cells stably transfected with either empty vector (ctr) or the same vector encoding human p66Shc (p66) are shown on the right. Stripped filters were reprobed with anti-actin antibodies as loading control. (C) Quantification by real-time RT-PCR of the levels of p66Shc mRNA in peripheral blood B cells purified from 21 healthy donors. Transcript levels were normalized to the expression level of GAPDH. For each donor, Syber 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 ΔΔCT method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on donor C1. The histograms show the mean ± SD of the 4 reactions carried out for each RNA. Statistical analysis (Student t test) did not show any significant difference in p66Shc mRNA levels among healthy donors. (D) Flow cytometric analysis of annexin V staining on Ramos B cells stably transfected with either empty vector (ctr) or the same vector encoding human p66Shc (p66), activated for 24 and 48 hours with anti-IgM antibodies. The graph shows the percentage (mean value ± SD) of annexin V+ cells (n = 3). (E) Flow cytometric analysis of annexin V staining of splenocytes from control (+/+) or p66Shc−/− (−/−) mice activated for 24 and 48 hours with goat IgG fraction specific for mouse immunoglobulins (40 μg/mL). The analysis was carried out on gated CD22+ cells. The graph shows the percentage (mean value ± SD) of annexin V+ splenocytes (n = 4; ***P ≤ .001, **P ≤ .01, *P ≤ .05).$

p66Shc acts as a negative regulator of B-cell survival. (A) Detection of p66Shc-specific mRNA in peripheral blood B cells purified from healthy donors by semiquantitative RT-PCR. The identity of the band was confirmed by automated sequencing. (B) Immunoblot analysis of p66Shc expression in lysates of peripheral blood B cells purified from 4 representative healthy donors. Lysates of Ramos B cells stably transfected with either empty vector (ctr) or the same vector encoding human p66Shc (p66) are shown on the right. Stripped filters were reprobed with anti-actin antibodies as loading control. (C) Quantification by real-time RT-PCR of the levels of p66Shc mRNA in peripheral blood B cells purified from 21 healthy donors. Transcript levels were normalized to the expression level of GAPDH. For each donor, Syber 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 donor C1. The histograms show the mean ± SD of the 4 reactions carried out for each RNA. Statistical analysis (Student t test) did not show any significant difference in p66Shc mRNA levels among healthy donors. (D) Flow cytometric analysis of annexin V staining on Ramos B cells stably transfected with either empty vector (ctr) or the same vector encoding human p66Shc (p66), activated for 24 and 48 hours with anti-IgM antibodies. The graph shows the percentage (mean value ± SD) of annexin V⁺ cells (n = 3). (E) Flow cytometric analysis of annexin V staining of splenocytes from control (+/+) or p66Shc^−/− (−/−) mice activated for 24 and 48 hours with goat IgG fraction specific for mouse immunoglobulins (40 μg/mL). The analysis was carried out on gated CD22⁺ cells. The graph shows the percentage (mean value ± SD) of annexin V⁺ splenocytes (n = 4; ***P ≤ .001, **P ≤ .01, *P ≤ .05).

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