American Society of Hematology

Figure 4.

$Figure 4. PILSAP binds and modifies PDK1 allowing the association of S6K. (A) MSS31 cells were transfected with an expression plasmid encoding myc-tagged PDK1. Cells were treated with or without VEGF (50 ng/mL). Cell lysates were immunoprecipitated (IP) with anti-myc antibody (Ab). Immunoprecipitates were resolved and immunoblotted (IB) with the indicated antibodies. PDK1 binds under basal conditions, and S6K associates with these proteins upon VEGF stimulation. (B) MSS31 cells were transfected with the PDK1 expression vector and stained with an anti-His (PDK1) or anti-PILSAP antibody. Immunofluorescent staining of PDK1 (green) and PILSAP (red) was performed. Cells were observed by confocal microscopy (LSM410; Carl Zeiss, Jena, Germany). Objective lens: Plan Neofluor 20×; camera: 35 mm Contax 167MT; software: LSM3.98. (C) COS-7 cells were transfected with the PILSAP expression vector and the subcellular localization of PILSAP was analyzed as described in “Materials and methods.” Lane 1 shows the total; lane 2, cytosolic fraction; lane 3, membrane fraction; and lane 4, microsomal fraction. (D) After treatment with ODNs or with LT, MSS31 cells were serum starved for 24 hours and then stimulated with VEGF (50 ng/mL) for 15 minutes. Cell lysates were immunoprecipitated with an anti-PDK1Ab. Precipitates were then immunoblotted with anti-PDK1 and anti-PILSAP Abs or with anti-PDK1 and anti-S6K Abs. (E) HEK293 cells were transfected with a PDK1-myc expression vector and cell lysates were incubated with purified GST-tagged PILSAP constructs (H1, H2, T1, T2) for pull-down analysis. (F) Purified PILSAP and PDK1 were incubated for 30 minutes at 37° C and subjected to immunoblotting of PDK1. The N-terminal amino acid sequence was determined. *The cleaved PDK1.$

PILSAP binds and modifies PDK1 allowing the association of S6K. (A) MSS31 cells were transfected with an expression plasmid encoding myc-tagged PDK1. Cells were treated with or without VEGF (50 ng/mL). Cell lysates were immunoprecipitated (IP) with anti-myc antibody (Ab). Immunoprecipitates were resolved and immunoblotted (IB) with the indicated antibodies. PDK1 binds under basal conditions, and S6K associates with these proteins upon VEGF stimulation. (B) MSS31 cells were transfected with the PDK1 expression vector and stained with an anti-His (PDK1) or anti-PILSAP antibody. Immunofluorescent staining of PDK1 (green) and PILSAP (red) was performed. Cells were observed by confocal microscopy (LSM410; Carl Zeiss, Jena, Germany). Objective lens: Plan Neofluor 20×; camera: 35 mm Contax 167MT; software: LSM3.98. (C) COS-7 cells were transfected with the PILSAP expression vector and the subcellular localization of PILSAP was analyzed as described in “Materials and methods.” Lane 1 shows the total; lane 2, cytosolic fraction; lane 3, membrane fraction; and lane 4, microsomal fraction. (D) After treatment with ODNs or with LT, MSS31 cells were serum starved for 24 hours and then stimulated with VEGF (50 ng/mL) for 15 minutes. Cell lysates were immunoprecipitated with an anti-PDK1Ab. Precipitates were then immunoblotted with anti-PDK1 and anti-PILSAP Abs or with anti-PDK1 and anti-S6K Abs. (E) HEK293 cells were transfected with a PDK1-myc expression vector and cell lysates were incubated with purified GST-tagged PILSAP constructs (H1, H2, T1, T2) for pull-down analysis. (F) Purified PILSAP and PDK1 were incubated for 30 minutes at 37° C and subjected to immunoblotting of PDK1. The N-terminal amino acid sequence was determined. *The cleaved PDK1.

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