Figure 1.
Figure 1. BCL11B up-regulates the levels of IL-2 mRNA in transformed and primary CD4+ T lymphocytes. (A, top) Expression of BCL11B evaluated by qRT-PCR in primary CD4+ T cells in the following conditions: freshly isolated (naive), stimulated for 24 hours with anti-CD3/anti-CD28 (activated), rested for 72 hours following the anti-CD3/anti-CD28 treatment (resting), or rested and restimulated with 50 ng/mL PMA and 1 μM ionomycin for 4 hours (restimulated). The relative abundance of BCL11B message was normalized to actin and calculated as described in “Reverse transcription and real-time PCR—quantitative RT-PCR (qRT-PCR).” (Bottom) Western blot analysis of BCL11B in primary CD4+ T cells treated in the conditions shown in the top panel; BCL11B levels were quantified by densitometric analysis and normalized to actin. (B) BCL11B expression evaluated by RT-PCR (top) and Western blot analysis (bottom) in Jurkat cells grown in the presence or absence of PMA (50 ng/mL) and ionomycin (1 μM) for 4 hours. BCL11B levels were quantified by densitometric analysis and normalized to HDAC2. (C) Schematic representation of the MSCV and MSCV-BCL11B retroviral constructs. LTR represents long terminal repeat; IRES, internal ribosomal entry site. (D) Western blot analysis demonstrating expression of Flag-BCL11B in retrovirally transduced Jurkat cells. (E) Levels of IL-2 mRNA evaluated by RT-PCR (top) and qRT-PCR (bottom) before (bottom) and after (both subpanels) treatment for 4 hours with PMA/ionomycin in MSCV and MSCV-BCL11B Jurkat cells. (F) Secretion of IL-2 determined by ELISA in media from MSCV and MSCV-BCL11B Jurkat cells after treatment with PMA/ionomycin for 16 hours. (G) Levels of IL-2 mRNA in MSCV and MSCV-BCL11B primary mouse CD4+ T cells after retroviral transduction and sorting of populations of GFP-positive cells. Cells were stimulated with PMA/ionomycin for 5 hours and the levels of IL-2 message were evaluated by qRT-PCR. The relative abundance of IL-2 message was normalized to actin and calculated as described in “Reverse transcription and real-time PCR—quantitative RT-PCR (qRT-PCR).” Three to 5 experiments were conducted, and the quantifications represent means ± SD.

BCL11B up-regulates the levels of IL-2 mRNA in transformed and primary CD4+ T lymphocytes. (A, top) Expression of BCL11B evaluated by qRT-PCR in primary CD4+ T cells in the following conditions: freshly isolated (naive), stimulated for 24 hours with anti-CD3/anti-CD28 (activated), rested for 72 hours following the anti-CD3/anti-CD28 treatment (resting), or rested and restimulated with 50 ng/mL PMA and 1 μM ionomycin for 4 hours (restimulated). The relative abundance of BCL11B message was normalized to actin and calculated as described in “Reverse transcription and real-time PCR—quantitative RT-PCR (qRT-PCR).” (Bottom) Western blot analysis of BCL11B in primary CD4+ T cells treated in the conditions shown in the top panel; BCL11B levels were quantified by densitometric analysis and normalized to actin. (B) BCL11B expression evaluated by RT-PCR (top) and Western blot analysis (bottom) in Jurkat cells grown in the presence or absence of PMA (50 ng/mL) and ionomycin (1 μM) for 4 hours. BCL11B levels were quantified by densitometric analysis and normalized to HDAC2. (C) Schematic representation of the MSCV and MSCV-BCL11B retroviral constructs. LTR represents long terminal repeat; IRES, internal ribosomal entry site. (D) Western blot analysis demonstrating expression of Flag-BCL11B in retrovirally transduced Jurkat cells. (E) Levels of IL-2 mRNA evaluated by RT-PCR (top) and qRT-PCR (bottom) before (bottom) and after (both subpanels) treatment for 4 hours with PMA/ionomycin in MSCV and MSCV-BCL11B Jurkat cells. (F) Secretion of IL-2 determined by ELISA in media from MSCV and MSCV-BCL11B Jurkat cells after treatment with PMA/ionomycin for 16 hours. (G) Levels of IL-2 mRNA in MSCV and MSCV-BCL11B primary mouse CD4+ T cells after retroviral transduction and sorting of populations of GFP-positive cells. Cells were stimulated with PMA/ionomycin for 5 hours and the levels of IL-2 message were evaluated by qRT-PCR. The relative abundance of IL-2 message was normalized to actin and calculated as described in “Reverse transcription and real-time PCR—quantitative RT-PCR (qRT-PCR).” Three to 5 experiments were conducted, and the quantifications represent means ± SD.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal