American Society of Hematology

Figure 2

$Figure 2. Role of Bim up-regulation in tipifarnib-induced apoptosis. (A-B) After Jurkat cells were treated for 72 hours with the indicated tipifarnib concentration in the presence of 5μM Q-VD-OPh, a broad-spectrum caspase inhibitor, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigens. Hsp90 and β-actin served as loading controls. The shift in mobility of the farnesyltransferase substrate HDJ-2 confirmed the inhibition of farnesylation. In this and subsequent figures, gray and black arrows indicate farnesylated and unfarnesylated species, respectively. Asterisk (*) represents nonspecific band present in all lanes. (C) After Jurkat cells were treated with diluent (0.1% DSMO, −) or 800nM tipifarnib (+) in the presence Q-VD-OPh for 72 hours, the indicated subcellular fractions were isolated and subjected to immunoblotting. GAPDH served as a marker for cytosol; and cytochrome c oxidase subunit IV (CoX IV) served as a marker for mitochondria. (D) After Jurkat cells were treated with the indicated concentration of tipifarnib for 72 hours in the presence 5μM Q-VD-OPh, cell lysates prepared in 1% CHAPS37 were subjected to immunoprecipitation with protein G-Sepharose cross-linked to antibodies that recognize Bcl-2, Bcl-xL, or Mcl-1. Immunoprecipitates were washed, resolved by SDS-PAGE, and subjected to immunoblotting as indicated. In each immunoprecipitation experiment, tubes lacking primary antibody served as controls to assess the specificity of protein recovery with the protein G-Sepharose beads. (E) Twenty-four hours after transfection of control oligonucleotide or Bim siRNA along with plasmid encoding EGFP-histone H2B (to mark successfully transfected cells), cells were treated for 48 hours with tipifarnib before staining with APC-conjugated annexin V and analysis by 2-color flow cytometry. Error bars indicate mean ± SD of 3 experiments. (E) Inset: Immunoblots of whole cell lysates prepared from siRNA-treated cells incubated in drug-free medium in parallel with samples harvested for flow cytometry.$

Role of Bim up-regulation in tipifarnib-induced apoptosis. (A-B) After Jurkat cells were treated for 72 hours with the indicated tipifarnib concentration in the presence of 5μM Q-VD-OPh, a broad-spectrum caspase inhibitor, whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies that recognize the indicated antigens. Hsp90 and β-actin served as loading controls. The shift in mobility of the farnesyltransferase substrate HDJ-2 confirmed the inhibition of farnesylation. In this and subsequent figures, gray and black arrows indicate farnesylated and unfarnesylated species, respectively. Asterisk (*) represents nonspecific band present in all lanes. (C) After Jurkat cells were treated with diluent (0.1% DSMO, −) or 800nM tipifarnib (+) in the presence Q-VD-OPh for 72 hours, the indicated subcellular fractions were isolated and subjected to immunoblotting. GAPDH served as a marker for cytosol; and cytochrome c oxidase subunit IV (CoX IV) served as a marker for mitochondria. (D) After Jurkat cells were treated with the indicated concentration of tipifarnib for 72 hours in the presence 5μM Q-VD-OPh, cell lysates prepared in 1% CHAPS³⁷ were subjected to immunoprecipitation with protein G-Sepharose cross-linked to antibodies that recognize Bcl-2, Bcl-x_L, or Mcl-1. Immunoprecipitates were washed, resolved by SDS-PAGE, and subjected to immunoblotting as indicated. In each immunoprecipitation experiment, tubes lacking primary antibody served as controls to assess the specificity of protein recovery with the protein G-Sepharose beads. (E) Twenty-four hours after transfection of control oligonucleotide or Bim siRNA along with plasmid encoding EGFP-histone H2B (to mark successfully transfected cells), cells were treated for 48 hours with tipifarnib before staining with APC-conjugated annexin V and analysis by 2-color flow cytometry. Error bars indicate mean ± SD of 3 experiments. (E) Inset: Immunoblots of whole cell lysates prepared from siRNA-treated cells incubated in drug-free medium in parallel with samples harvested for flow cytometry.

This Feature Is Available To Subscribers Only