Pgex kg

For the eukaryotic expression vectors encoding FLAG-, MYC-fusion proteins tagged at the amino terminus were constructed by inserting PCR-amplified fragments into pcDNA3 (Invitrogen) or pIRESpuro2 (Clontech). LEF1-response element (LRE) promoter luciferase reporters were generated through insertion of PCR-amplified promoter fragments from genomic DNA into the pGL4-basic vector (Promega). Lentiviral shRNA vectors were generated after cloning short hairpin RNA (shRNA) fragments into the lentiviral vector pSIH-H1-Puro (System Biosciences). Lentiviruses were produced through cotransfection of HEK293T cells (RRID:CVCL_0063) with recombinant lentivirus vectors and the pPACK Packaging Plasmid Mix (System Biosciences) using a Megatran reagent (Origene). Lentiviruses were collected at 48 h after transfection and added to the medium of target cells with 8 μg/ml Polybrene (Sigma-Aldrich). Plasmids encoding GST-fusion proteins were prepared by cloning into pGEX-KG (Amersham Pharmacia Biotech). For the yeast two-hybrid assay, the bait plasmid pAS2-LEF1 was constructed by inserting the full-length LEF1 cDNA fragment into pAS2-1 (Clontech). Details of the cloning are available upon request.

The eukaryotic expression constructs for GATA1 were generated by cloning PCR-amplified full length sequences into pcDNA3 (Invitrogen). GST-fusion protein encoding vectors were constructed by inserting PCR-amplified sequences into pGEX-KG (Amersham Pharmacia Biotech). The Bcl-XL promoter luciferase reporters were obtained by cloning PCR-amplified promoter fragments into pGL4-basic vector (Promega). The mutated Bcl-XL promoter luciferase reporters were constructed by recombinant PCR. Lentiviral vector of GATA1 was constructed by cloning PCR-amplified full length sequences into pCDH (System Biosciences). The short hairpin RNAs (shRNAs) targeting GATA1 and Bcl-XL cDNA were inserted into PSIH-H1-puro (System Biosciences). The small interfering RNAs sharing the same targets with shRNAs were synthetized from GenePharma (Shanghai, China). The sequences for siRNAs and shRNAs are listed in Table S4a.
Specific antibodies against GATA1 (ab28839) and Bax (ab32503) were purchased from Abcam. Anti-Bcl-XL (#2764), anti-cleaved PARP (#5625), anti-cleaved caspase 3 (#9664), and anti-cleaved caspase 9 (#9505) were from Cell Signaling Technology. Anti-β-actin (sc-47778HRP) was from Santa Cruz Biotechnology. Gemcitabine (T0251) was obtained from Targetmol.