Nkx6.2 synergizes with Cdx-2 in stimulating proglucagon gene expression

Figure 1 The sliver staining of SDS-PAGE for eluates from GST or GST-Cdx-2 column with cell lysate of GLUTag or InR1-G9. GSH: Elution was performed by a buffer containing 50 mmol/L glutathione; SDS: Elution was performed by 1 × Laemmli SDS containing sample buffer. The arrows indicate the position of Nkx6.2. GST: the control GST protein; Cdx: Cdx-2/GST fusion protein.

Figure 2 Detection of Nkx6. 2 expression. A: Detection of Nkx6.2 mRNA expression by RT-PCR in pancreatic and intestinal endocrine cell lines. Detection of actin expression serves as the control for the quality of cDNA generated from each sample; B: Comparison of the expression of Nkx6.2 by real time RT-PCR in GLUTag cell line and the FRIC culture (n = 4).

Figure 3 The interaction between Cdx-2 and Nkx6. 2. A: Purification of GST and GST-Nkx6.2 fusion protein from bacteria lysate. Lane 1: GST; Lane 2: GST-Nkx6.2; M: molecular weight marker; B1: Left panel. GST-Cdx-2 pulls down Nkx6.2 but not Nkx6.1. GST-Cdx-2 column was incubated with lysate either from pcDNA3-Nkx6.1-myc transfected BHK cells (lanes 1, 2 and 3) or from pcDNA3-Nkx6.2-myc transfected cells (lanes 4, 5 and 6). Myc tag antibody was utilized in Western blotting; B2: Right panel. GST-Nkx6.2 pulls down Cdx-2. GST-Nkx6.2 column was incubated with cell lysate from pcDNA3-Cdx-2-myc transfected BHK cells.

Figure 4 Effect of ectopic Nkx6. 2 expression on endogenous Gcg expression in the In111 and IEC6 cells. A: Transient transfection of Cdx-2, Nkx6.1 and Nkx6.2 in In111 cells. Cell lysates from In111 cells transfected with Myc-taged Cdx-2, Nkx6.1 and Nkx6.2 was prepared and analyzed for their expression by Western blotting. The same membrane was stripped and re-hybridized against the β-actin antibody; B: The expression of endogenous Gcg mRNA was assayed by real time RT-PCR. The Gcg mRNA expression was quantified against that of actin; C: Transient transfection of Nkx6.2 (by anti-Myc tag antibody) and Cdx-2 (by anti-Cdx-2 antibody) in the IEC6 cell line; D: The expression of Gcg mRNA was assayed by real time RT-PCR.