Figure 1
Figure 1. TGF-β induces the proteasome-dependent degradation of TAL1. (A) Decreased expression of TAL1 in the presence of TGF-β. Jurkat cells were treated with 10 ng/mL TGF-β during indicated times, and extracts were prepared from these cells were analyzed by immunoblot using antibodies to TAL1 (top panel) or to β-actin, used here as loading control (bottom panel). (B) TAL1 transcription is not affected by TGF-β. Total RNAs from TGF-β–treated Jurkat cells were purified and analyzed by real-time quantitative RT-PCR for TAL1 mRNA. Quantification was performed with respect to untreated cells and normalized by β-actin mRNA. Mean of 3 measures is represented with standard deviation as percentage with respect to untreated cells. (C) MG132 reverses TGF-β-induced degradation of TAL1. HeLa cells transfected with pSGF-TAL110 (lanes 1 to 3) or Jurkat cells (lanes 4-6) were treated with 10 ng/mL of TGF-β during 9 hours and with or without addition of 10 μM MG132 during the last 6 hours. Cellular extracts were normalized with respect to protein concentration and analyzed by immunoblot using antibodies to TAL1 (top panel) or to β-actin (bottom panel). (D) TGF-β-induced degradation of TAL1 in leukemic cells. Either fresh or frozen cells from 5 patients suffering T-ALL with TAL1 deregulation (4 SIL-TAL1 rearrangement and one translocation t(1;14)) were cultured in RPMI 1640 plus 10% fetal calf serum and were treated without or with 15, 30, 45, 60, and 75 ng/mL TGF-β for 6 hours. Extracts were prepared in radioimmunoprecipitation assay buffer and equal total protein amounts were analyzed by immunoblot as described for panel C. Results from one representative patient are shown. (E) TAL1 is polyubiquitylated. HeLa cells were transfected with 1 μg of pSGF-TAL1 and 0.5 μg of pSG-HA-Ub,12 with or without MG132 treatment as indicated. Cell lysates were immunoprecipitated with an antibody to FLAG. Immunoblot analysis of extracts (lanes 1-3) was done using the antibody to HA and that of immunoprecipitates (lanes 4-6) using antibodies to HA (top panel) and FLAG (bottom panel).

TGF-β induces the proteasome-dependent degradation of TAL1. (A) Decreased expression of TAL1 in the presence of TGF-β. Jurkat cells were treated with 10 ng/mL TGF-β during indicated times, and extracts were prepared from these cells were analyzed by immunoblot using antibodies to TAL1 (top panel) or to β-actin, used here as loading control (bottom panel). (B) TAL1 transcription is not affected by TGF-β. Total RNAs from TGF-β–treated Jurkat cells were purified and analyzed by real-time quantitative RT-PCR for TAL1 mRNA. Quantification was performed with respect to untreated cells and normalized by β-actin mRNA. Mean of 3 measures is represented with standard deviation as percentage with respect to untreated cells. (C) MG132 reverses TGF-β-induced degradation of TAL1. HeLa cells transfected with pSGF-TAL110  (lanes 1 to 3) or Jurkat cells (lanes 4-6) were treated with 10 ng/mL of TGF-β during 9 hours and with or without addition of 10 μM MG132 during the last 6 hours. Cellular extracts were normalized with respect to protein concentration and analyzed by immunoblot using antibodies to TAL1 (top panel) or to β-actin (bottom panel). (D) TGF-β-induced degradation of TAL1 in leukemic cells. Either fresh or frozen cells from 5 patients suffering T-ALL with TAL1 deregulation (4 SIL-TAL1 rearrangement and one translocation t(1;14)) were cultured in RPMI 1640 plus 10% fetal calf serum and were treated without or with 15, 30, 45, 60, and 75 ng/mL TGF-β for 6 hours. Extracts were prepared in radioimmunoprecipitation assay buffer and equal total protein amounts were analyzed by immunoblot as described for panel C. Results from one representative patient are shown. (E) TAL1 is polyubiquitylated. HeLa cells were transfected with 1 μg of pSGF-TAL1 and 0.5 μg of pSG-HA-Ub,12  with or without MG132 treatment as indicated. Cell lysates were immunoprecipitated with an antibody to FLAG. Immunoblot analysis of extracts (lanes 1-3) was done using the antibody to HA and that of immunoprecipitates (lanes 4-6) using antibodies to HA (top panel) and FLAG (bottom panel).

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