a. Expression of TBLR1 in mouse tissues (Western Blotting). Anti peptide antibodies were made against deduced sequences from TBLR1. The antibody used was prepared against a sequence unique to TBL1 does not react with TBL1. After transfer, the membranes were stained with polyclonal rabbit anti-TBLR1 peptide antibodies and the transferred proteins visualized with HRP-labeled donkey anti-rabbit IgG. The bound HRP was detected using luminol as the substrate. b. Expression of TBLR1 by HK293T cells (Western Blotting). TBLR1 proteins were isolated by immuno-affinity chromatography from lysates of HK293T cells. Rabbit anti-TBLR1(117-125), covalently coupled to the gel, was used as the immunoadsorbant. The bound material was eluted with a glycine-HCl buffer (pH 2.8) and then run on the gels. After transfer to nitrocellulose, these were stained with either anti TLR1α or TRLR1β or with anti anti-TBLR1(117-125) which detects both isoforms. The proteins were visualized as above. c. Immunohistochemical Detection of the expression of TBLR1 in human fetal liver. All of the samples were from 17 week fetuses. The sample on the left was stained with the anti-TBLR1 antiserum that detects both isoforms but does not detect TBL1. The staining was detected with HRP coupled goat anti rabbit IgG using DAB as the chromagen. The original magnification was 40×. A digital image was captured and cropped in Photoshop. The images on the right show samples stained with the specific anti α and β sera. They were also detected with HRP coupled goat anti rabbit IgG but using AEC as a chromagen. The images were captured on film and then scanned to produce the digital images shown. The original magnification was 40×. The hematopoietic cells of the fetal liver stained more intensely with the anti TBLR1β antiserum. All of the samples were counterstained with haematoxylin.
Zhang et al. BMC Cell Biology 2006 7:31 doi:10.1186/1471-2121-7-31