human islet and brain cdna  (OriGene)

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet ?-cells

doi: 10.1152/ajpendo.00664.2009

Figure Lengend Snippet: Expression of neuronal adaptor protein (AP)-3 mRNA and protein in INS-1 β-cells and pancreatic islet extracts. A: PCR analysis was performed to determine whether the neuronal AP-3 subunits β3B and μ3B are expressed in human islets (Isl). Human brain (Br) cDNA was used as a positive control. PCR products of the expected size were detected in brain and islets for both β3B and μ3B. A negative control (−) in which no reverse transcriptase was added during the reverse transcription reaction (no RT control) is shown to the right of the brain and islet lanes. cDNA from human tissues (shown) and from rat tissues (not shown) yielded identical results. B: to determine whether β3B protein is expressed in islets, Western blot analysis was performed using a β3B-specific monoclonal antibody. As expected, β3B was detected in human (H), rat (R), and mouse (M) Br. β3B protein was also present in cell lysates from the insulin-secreting β-cell lines β-TC3 (β), HIT-T15 (Hit), and INS-1 (Ins) and in tissue lysates from human and rat Isl. β3B was not detected in human liver or in brain from β3B-knockout mice (Ref. 43 and data not shown). As observed previously, immunoblot detection of β3B yielded 2 bands (Mr of ∼140 K). C, left, lanes 1 and 2: μ3B was detected in INS-1 cell lysate (Ins) but not in rat liver lysate (Liv). C, right: μ3B produced by in vitro transcription and translation (lane 4) and analyzed by immunoblotting with the μ3B antibody ran at the expected Mr of ∼47,000. This band comigrated precisely with the band that was observed in INS-1 cells (lane 3). 35S-met was included in the in vitro translation reaction. Autoradiographic detection of the radiolabeled μ3B (lane 4) yielded a band that precisely aligned with the band detected by immunoblot analysis. This confirms that the μ3B antibody detected μ3B synthesized de novo in the in vitro transcription and translation reaction.

Article Snippet: Gene-specific primers were designed over exon-exon boundaries to the neuronal AP-3 subunits β3B (5′-AGCCAAGCTCTACCTGACCA-3′ and 5′-TCCAAGACTGGAGCTGGTTT-3′) and μ3B (5′-TGTCAGCTTCCATCCTTGTG-3′ and 5′-TCCCACCGTTATTTCAAAGC-3′), and PCR was performed using human islet and brain cDNA (OriGene Technologies, Rockville, MD).

Techniques: Expressing, Positive Control, Negative Control, Reverse Transcription, Control, Western Blot, Knock-Out, Produced, In Vitro, Synthesized