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Published in final edited form as: Exp Physiol. 2010 Dec 17;96(3):305–316. doi: 10.1113/expphysiol.2010.055038

A. Immuno-blots of the membrane fraction from distal colonic epithelial cells with anti-Tmem16A(Ano1) exhibited bands at 85kDa and 146kDa (arrowheads). These bands were not apparent with the secondary antibody alone, indicating that the primary antibody was necessary to observe results. A cell lysate of guinea pig brain exhibited a band at 84kDa. B. RT-PCR of mucosal mRNA amplified Tmem16A(Ano1) products with sizes predicted by the position of the primers (asterisks), 504 base pairs for the segment including exon-6B and 395-base-pairs for the segment omitting exon-15. The faint exon-6B product likely represented a splice variant lacking exon-6B, 438bp; and, the faint exon-15 product likely represented a splice variant including exon-15, 473bp. Sequencing of products confirmed identity with Tmem16A (homology: 87% human, 88% mouse, 87% rat). Amplification of GAPDH product (555bp) served as positive control for RNA isolation. Absence of product when not including reverse transcriptase indicated the lack of genomic DNA contamination. C. The sequence of the insert designated as variant-b (Ferrera etal., 2009) is labeled exon-6B and shown for rat, mouse, human, and guinea pig, with differences from human indicated by shading. D. In mucosa and epithelial cell samples from 4 colons, the dominant Tmem16A product from exon-6 to exon-16 (~770bp, arrowhead) was consistent with a transcript including exon-6B and exon-13 while omitting exon-15 (confirmed by sequencing; GenBank accession HQ341643). A smaller faint band (~710bp, asterisk) was consistent with a transcript including exon-13 and omitting both exon-6B and exon-15, and was 11±2% (n=4) of the total product in mucosa and 1±1% (n=3) of the total in epithelial cells. Markers were 900, 800, 766, and 600 base pairs. E. The exons of Tmem16A are shown schematically (alternatively spliced exons above), with the transcript in guinea pig distal colonic epithelial cells including exon-6B and exon-13 while omitting exon-15. The sequenced portion spans exon-4 to exon-16.

A. Immuno-blots of the membrane fraction from distal colonic epithelial cells with anti-Tmem16A(Ano1) exhibited bands at 85kDa and 146kDa (arrowheads). These bands were not apparent with the secondary antibody alone, indicating that the primary antibody was necessary to observe results. A cell lysate of guinea pig brain exhibited a band at 84kDa. B. RT-PCR of mucosal mRNA amplified Tmem16A(Ano1) products with sizes predicted by the position of the primers (asterisks), 504 base pairs for the segment including exon-6B and 395-base-pairs for the segment omitting exon-15. The faint exon-6B product likely represented a splice variant lacking exon-6B, 438bp; and, the faint exon-15 product likely represented a splice variant including exon-15, 473bp. Sequencing of products confirmed identity with Tmem16A (homology: 87% human, 88% mouse, 87% rat). Amplification of GAPDH product (555bp) served as positive control for RNA isolation. Absence of product when not including reverse transcriptase indicated the lack of genomic DNA contamination. C. The sequence of the insert designated as variant-b (Ferrera etal., 2009) is labeled exon-6B and shown for rat, mouse, human, and guinea pig, with differences from human indicated by shading. D. In mucosa and epithelial cell samples from 4 colons, the dominant Tmem16A product from exon-6 to exon-16 (~770bp, arrowhead) was consistent with a transcript including exon-6B and exon-13 while omitting exon-15 (confirmed by sequencing; GenBank accession HQ341643). A smaller faint band (~710bp, asterisk) was consistent with a transcript including exon-13 and omitting both exon-6B and exon-15, and was 11±2% (n=4) of the total product in mucosa and 1±1% (n=3) of the total in epithelial cells. Markers were 900, 800, 766, and 600 base pairs. E. The exons of Tmem16A are shown schematically (alternatively spliced exons above), with the transcript in guinea pig distal colonic epithelial cells including exon-6B and exon-13 while omitting exon-15. The sequenced portion spans exon-4 to exon-16.