They Didn't Believe That I Possibly Could Become A Venetoclax Pro...Today I Am!!

2.7. Western blotting Methods for Western blotting against either whole cell lysate or isolated mouse islets have been described in our previous studies [37,38]. 2.8. RNA isolation, reverse transcription, and quantitative RT-PCR RNA isolation, cDNA synthesis, and real-time RT-PCR (qPCR) were performed as previously described [39]. All nucleotide primers for qPCR are listed in Supplementary Table?1. 2.9. Statistical analysis Data are presented as means?��?SEM. Significance was determined using the Student's t-test or one-way ANOVA followed by Bonferroni post hoc test as appropriate for single or multiple comparisons respectively. Differences were considered statistically significant when p?Venetoclax cell line gene expression in the Ins-1 cell line Considering that other Tcfs, including Tcf7 and Tcf7l1, are also expressed in pancreatic ��-cells [40], which may exert redundant function with Tcf7l2, we tested the effect of functional knockdown of Tcfs or cat/Tcf with Ibrutinib supplier TCF7L2DN. The full-length human TCF7L2 cDNA (encoding the 78?kDa isoform) and one that lacks the first 117 bp nucleotide sequence (Figure?1A) were utilized to generate adenoviruses, designated as Ad-TCF7L2WT and Ad-TCF7L2DN. We reported very recently that TCF7L2DN is still able to bind to the consensus TCF binding motif but cannot recruit ��-cat [35]. When Ins-1 cells were infected with the GFP control virus (Ad-GFP), Ad-TCF7L2WT, or Ad-TCF7L2DN, the infection efficiency reached more than 95%, based on the detection of GFP expression (data not shown). The expression of endogenous Tcf7l2 and exogenous TCF7L2 can be detected by Western blotting with the TCF7L2 antibody, while TCF7L2 and TCF7L2DN can be differentiated based on their sizes (78?kDa versus 75?kDa; Figure?1B). The expression of exogenous TCF7L2 can be detected by the HA-tag antibody (Figure?1B). Ad-TCF7L2DN infection, but not Ad-TCF7L2WT infection, completely blocked GSIS (Figure?1C) and attenuated cell growth (Figure?1D). Wild type MMP23B TCF7L2-stimulated growth of Ins-1 cells was observed 48?h and 72?h after the infection (Figure?1D). Figure?1 TCF7L2DN expression in the Ins-1 cell line attenuates GSIS, inhibits cell growth and represses the expression of a battery of ��-cell specific genes. A) A schematic of TCF7L2DN (75?kDa) and wild type TCF7L2 (78?kDa). ��-cat, ... We then assessed the effect of Ad-TCF7L2WT and Ad-TCF7L2DN on the expression of two pro-insulin genes, genes that encode the two incretin hormone receptors (Gipr and Glp-1r), the Wnt pathway downstream target Axin2, and the ��-cell specific genes Mafa, Isl-1 and Pdx-1. As shown in Figure?1E, the expression of these genes was significantly inhibited by TCF7L2DN expression. Ad-TCF7L2WT infection generated no significant effect on the two pro-insulin genes and Pdx-1, increased the expression of the two incretin hormone receptors (Glp-1r and Gipr) and elevated the expression of Mafa and Isl-1.