Figure 1
Figure 1. Tcl1 interacts with Atm, and both affect IκBα expression. (A) HEK293 cells were stably transfected with expression plasmid encoding FLAG-ATM and then infected with Ad-TCL1 wild-type (MOI 100). Forty-eight hours after infection, whole-cell lysates were immunoprecipitated with anti-Atm (resin conjugated to Atm). The immunoprecipitates were analyzed by Western blot with anti-Atm or anti-Tcl1. Input: lysate expression of Atm (top) and Tcl1 (bottom). (B) HEK293 cells were transiently cotransfected with expression plasmids encoding mammalian FLAG-ATM (8 μg) and wild-type TCL1 (6 μg). Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-TCL1. The immunoprecipitates were analyzed by Western blot with anti-Atm or anti-Tcl1. Input: lysate expression of Atm (top) and Tcl1 (bottom). (C) HEK293 cells were transiently cotransfected with expression plasmids encoding mammalian Omni-GST-TCL1 (6 μg) or Omni-GST-FHIT (6 μg) and FLAG-HIS-ATM (8 μg). Forty-eight hours after transfection, cells were treated with neocarzinostatin (NCS) 200 ng/mL for 2 hours. Cell lysates were GST-pulled down and immunoblotted with anti-His6, anti-Tcl1, and anti-Fhit. Input: lysate expression of Atm. (D-E) Daudi cells were untreated or treated with hydroxyurea, 50mM for 3 hours, then lysed and immunoprecipitated, performed with anti-Atm, anti-Tcl1, or IgG. The precipitates were analyzed by Western blot with anti-Atm or anti-Tcl1. (F) Tcl1 and Atm coexpression is associated with decreased IκBα total protein. HEK293 cells were mock-transfected or transfected with TCL1 or ATM plasmids separately or together (4 μg for each plasmid, including empty vector). Forty-eight hours later, cells were treated with hydroxyurea 50mM for 3 hours and then analyzed by Western blot with the indicated antibodies. (G) Tcl1 and Atm coexpression is associated with increased IκBα (Ser32) phosphorylation as shown in the graph. (H) Daudi cells were mock-transfected or transfected with si-TCL1 or si-Scr, and then untreated or treated with Kudos 55933 or DMSO. The level of IκBα was measured by Western blot. (I) IκBα (Ser 32) levels are shown in the graph. Data are representative of 3 independent experiments.

Tcl1 interacts with Atm, and both affect IκBα expression. (A) HEK293 cells were stably transfected with expression plasmid encoding FLAG-ATM and then infected with Ad-TCL1 wild-type (MOI 100). Forty-eight hours after infection, whole-cell lysates were immunoprecipitated with anti-Atm (resin conjugated to Atm). The immunoprecipitates were analyzed by Western blot with anti-Atm or anti-Tcl1. Input: lysate expression of Atm (top) and Tcl1 (bottom). (B) HEK293 cells were transiently cotransfected with expression plasmids encoding mammalian FLAG-ATM (8 μg) and wild-type TCL1 (6 μg). Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-TCL1. The immunoprecipitates were analyzed by Western blot with anti-Atm or anti-Tcl1. Input: lysate expression of Atm (top) and Tcl1 (bottom). (C) HEK293 cells were transiently cotransfected with expression plasmids encoding mammalian Omni-GST-TCL1 (6 μg) or Omni-GST-FHIT (6 μg) and FLAG-HIS-ATM (8 μg). Forty-eight hours after transfection, cells were treated with neocarzinostatin (NCS) 200 ng/mL for 2 hours. Cell lysates were GST-pulled down and immunoblotted with anti-His6, anti-Tcl1, and anti-Fhit. Input: lysate expression of Atm. (D-E) Daudi cells were untreated or treated with hydroxyurea, 50mM for 3 hours, then lysed and immunoprecipitated, performed with anti-Atm, anti-Tcl1, or IgG. The precipitates were analyzed by Western blot with anti-Atm or anti-Tcl1. (F) Tcl1 and Atm coexpression is associated with decreased IκBα total protein. HEK293 cells were mock-transfected or transfected with TCL1 or ATM plasmids separately or together (4 μg for each plasmid, including empty vector). Forty-eight hours later, cells were treated with hydroxyurea 50mM for 3 hours and then analyzed by Western blot with the indicated antibodies. (G) Tcl1 and Atm coexpression is associated with increased IκBα (Ser32) phosphorylation as shown in the graph. (H) Daudi cells were mock-transfected or transfected with si-TCL1 or si-Scr, and then untreated or treated with Kudos 55933 or DMSO. The level of IκBα was measured by Western blot. (I) IκBα (Ser 32) levels are shown in the graph. Data are representative of 3 independent experiments.

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