Fig. 4

The CNVR_322 creates a ANXA10 null allele. a Schematic representation of exons 1 to 7 of ANXA10 (blue vertical bars) and the CNVR_322 region (magenta line). Primers for exons 1, 2, and 7 are represented by arrows. b PCR amplification of cDNA from the abomasum within and across the CNVR_322 region for four wild-type (lanes 1, 4, 5, 6) and two carrier (lanes 2, 3) animals. PCR primers for exons 1 and 2 amplified an allele (229 bp) in all animals (b1), and PCR primers for exons 1 and 7 amplified a wild-type allele (600 bp) and two mutant alleles (300 bp and 197 bp) (b2). M, 100 bp ladder markers. c The predicted amino acid (aa) length and molecular mass of wild-type (c1), exon 2 to 6 deleted-type (c2), and exon 2 to 6 deleted-/inserted-type (c3) is 324 aa (37 kDa), 170 aa (19.7 kDa), and 200 aa (22.3 kDa). In the exon 2 to 6 deleted-/inserted-type protein, 30 aa (bracket) was inserted in the 178 aa position (c3). d Western blotting for wild-type (lanes 1, 4, 5, 6) and two carrier (lanes 2, 3) animals using antibodies to full length (AF3544) and to the C terminal region (ab135985) of the ANXA10 protein. Recombinant bovine ANXA10 proteins were expressed in HeLa cells by pCAGGS-ANXA10 expression plasmid and used as a positive control.　A specific band was detected for ANXA10 at approximately 30 kDa (arrow). Brackets represent migration areas below 30 kDa. e Quantitation of ANXA10 bands (d2) were relative to a wild-type animal (lane 1). Relative ANXA10 band intensities were measured using the ImageQuant TL Analysis Toolbox. The amounts of loaded proteins were calibrated using Coomassie Brilliant Blue-stained bands (d1) between 25 and 50 kDa. The dashed line represents average intensities from wild-type (black) and CNVR_322 animals (magenta)