Pcdna3.1 flag expression vector

The human full-length cDNA of iASPP was cloned from pcDNA3.1-WT-iASPP-V5 (a kind gift from Prof. Lu Xin, Ludwig Institute for Cancer Research, Oxford, UK) into pcDNA3.1-Flag expression vector (Invitrogen, USA). Full-length cDNA and different truncation mutants of FHL2 were cloned from cDNA of HEK293T cells into pcDNA3.1-Myc expression vector (Invitrogen, USA). Plasmids were co-transfected into HEK293T cells using polyethylenimine (PEI) (Polyscience, USA). Specific primers for iASPP and FHL2 were synthesized by Invitrogen (Table 1). FHL2 specific (shFHL2) and scrambled control (SCR) shRNAs were designed by Ambion software. The target sequence of FHL2 was 5’-CGACTGCTTTAACTGTAAGAA-3’ and the scramble sequence was 5’-CAACAAGATGAAGAGCACCAA-3’. Fragments were inserted into the PLKO.1-GFP vector. Lentiviral vector constructs and packaging vectors (pCMV and pMDG) were co-transfected into HEK293T cells using PEI to produce lentivirus.

The coding sequence of wild-type or mutant Skp1 and NIPA was cloned into the pcDNA3.1-flag expression vector (Invitrogen, Carlsbad, CA, USA). The siRNAs targeting candidate genes were designed and synthesized by Shanghai GenePharma Co., the siRNA sequences were as follows: 5′-CGCAAGACCUUCAAUAUCATT-3′ (Skp1 siRNA1), 5′-CCAAUAUGAUCAAGGGGAATT-3′ (Skp1 siRNA2), and 5′-GUCCACGUCACUGCCUGUATT-3′ (NIPA siRNA). Using lipofectamine 2000 (Invitrogen, California, USA), A549 cells were transfected with 100 nM siRNA. And 48 h later, the cells were treated with or without 6-OAP at 7.5 μM for indicated time points. The cells were then harvested for cell cycle analysis, immunofluorescence staining, or lysed for Western blotting.

cDNA encoding human sMEK1 was introduced into pcDNA3.1/Flag expression vector (Invitrogen) and digested with EcoRI and XhoI (pcDNA3.1/Flag-sMEK1). The human VEGFR-1 and VEGFR-2 cDNA was ligated into pcDNA3.1 expression vector (Invitrogen) using EcoRI and XhoI (pcDNA3.1-VEGFR-1 and pcDNA3.1-VEGFR-2). For co-immunoprecipitation, HEK293T cells were co-transfected with cDNA constructs of pcDNA3.1/Flag-sMEK1 and pcDNA3.1-VEGFR-1 or pcDNA3.1-VEGFR-2 using Lipofectamine 2000 transfection reagent (Invitrogen). Cells were trypsinized and then centrifuged. Cell pellets were washed in PBS, resuspended in lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail containing 1 μg/ml leupeptin, 1 μg/ml pepstatin, 2 μg/ml aprotinin, 200 μg/ml PMSF). Lysates were then incubated with anti-Flag antibody (Santa Cruz) and precipitated with protein A-agarose (Amersham). The precipitated proteins were separated by SDS-PAGE electrophoresis, transferred onto Immobilon P membrane (Millipore corporation, Billerica, MA), and immunoblotted with anti-sMEK1, anti-VEGFR-1, or anti-VEGFR-2 antibody using the ECL system (Amersham).