mafa-specific antibody  (Bethyl)

Journal: The Journal of Biological Chemistry

Article Title: Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice *

doi: 10.1074/jbc.M116.770032

Figure Lengend Snippet: β-Cell dysfunction and aging in SMAD7Ptf1a mice is characterized by a gradual loss of β cell identity genes. A, RT-qPCR gene transcript enrichments for Pdx1, NeuroD1, MafA, and Nkx6.1 were analyzed in isolated islets from differently aged SMAD7Ptf1a and littermate control SMAD7fx/fx mice. The values were normalized against CycloA, which was consistent throughout all conditions. B, representative images of co-immunostaining for insulin (INS) with Pdx1, NeuroD1, MafA, and Nkx6.1, respectively. *, p < 0.05 and n = 5 in all cases. Scale bar = 50 μm.

Article Snippet: Primary antibodies were as follows: guinea pig polyclonal insulin-specific (Dako, Carpinteria, CA); goat polyclonal NeuroD1-specific (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal SMAD7-, CyclinD1-, and CyclinD2-specific (Santa Cruz Biotechnology); FoxO1- and GAPDH-specific (Cell Signaling Technology, San Jose, CA), Collagen I- and Pdx1-specific (Abcam, Cambridge, MA, USA), MafA-specific (Bethyl Laboratories, Inc.); Nkx6.1-specific (a kind gift from Dr. Maike Sander, University of California, San Diego, CA); and rat CD31-specific (BD Biosciences) and BrdU-specific (Abcam).

Techniques: Quantitative RT-PCR, Isolation, Control, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice *

doi: 10.1074/jbc.M116.770032

Figure Lengend Snippet: Forced expression of FoxO1, but not SMAD7 in β cells, inhibited β cell dysfunction and onset of diabetes in SMAD7Ptf1a mice. A, SMAD7Ptf1a mice received either AAV-RIP-FoxO1, AAV-RIP-SMAD7, or AAV-RIP-GFP as a control at 20 weeks of age when glucose intolerance was present. After viral infusion, the mice were followed for 10 more weeks (30 weeks of age). BrdU was given for the last week prior to harvest. IHC, immunohistochemistry. B, Western blotting on islets for FoxO1. w, weeks. C, Western blotting on islets for SMAD7. D, fasting blood glucose measurement of SMAD7Ptf1a mice after intraductal infusion of AAV-RIP-FoxO1, AAV-RIP-SMAD7, or control AAV-RIP-GFP. E, IPGTT of the SMAD7Ptf1a mice 10 weeks after viral infusion. F, messenger RNA was analyzed by RT-qPCR on islets at 30 weeks of age, showing a significant increase in NeuroD1, MafA, and Nkx6.1, but not Pdx1, and cell cycle activators specifically in AAV-RIP-FoxO1-infused pancreata. G, representative immunostaining images showed significant improvement in MafA and NeuroD1 signals in β cells from SMAD7Ptf1a mice that received FoxO1 virus. INS, insulin; HO, Hoechst. H, perfusion of isolated islets from the SMAD7Ptf1a mice 10 weeks after viral infusion. I and J, intraductal infusion of AAV-RIP-SMAD7, but not AAV-RIP-FoxO1, significantly increased β cell replication in SMAD7Ptf1a mice, shown by quantification (I) and by representative immunostaining images (J). *, p < 0.05 and n = 5 in all cases. Scale bars = 50 μm.

Article Snippet: Primary antibodies were as follows: guinea pig polyclonal insulin-specific (Dako, Carpinteria, CA); goat polyclonal NeuroD1-specific (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal SMAD7-, CyclinD1-, and CyclinD2-specific (Santa Cruz Biotechnology); FoxO1- and GAPDH-specific (Cell Signaling Technology, San Jose, CA), Collagen I- and Pdx1-specific (Abcam, Cambridge, MA, USA), MafA-specific (Bethyl Laboratories, Inc.); Nkx6.1-specific (a kind gift from Dr. Maike Sander, University of California, San Diego, CA); and rat CD31-specific (BD Biosciences) and BrdU-specific (Abcam).

Techniques: Expressing, Control, Immunohistochemistry, Western Blot, Quantitative RT-PCR, Immunostaining, Virus, Isolation