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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: J Immunol. 2012 Mar 5;188(7):3031–3041. doi: 10.4049/jimmunol.1103226

FIGURE 4.

FIGURE 4

Expression of Ly108 isoforms in C57Bl/6 and 129S6 thymocytes A. Schematic of Ly108 isoforms cloned from C57Bl/6 thymocytes. ITSMs at the carboxy-termini are indicated in hatched boxes and the non-classical ITSM is indicated by a grey box. Non-ITSM tyrosines are in black. The Ly108-1 isoform, has 2 classical ITSMS and 1 non-ITSM tyrosine, Y330. The Ly108-2 isoform, has 2 classical ITSMs, 1 non-classical ITSM (Y335) and 1 non-ITSM tyrosine (Y349). The novel Ly108-H1 isoform, has 1 classical ITSM (Y295), 1 non-classical ITSM (Y311) and 1 non-ITSM tyrosine (Y325), which is equivalent to Y349 of Ly108-2. White bars below exon 2 indicate the extracellular domain peptide used to generate pan-Ly108 polyclonal antibody that detects all the three isoforms. Grey bars below exon 9 indicate the location of the cytoplasmic peptide used to generate αLy108-2, which recognizes Ly108-2 and Ly108-H1, but does not react with Ly108-1. TM: transmembrane domain. B. Real time Taqman probes specific for Ly108-1, 2 and novel Ly108-H1 isoforms were used to detect expression in RNA isolated from sorted and DP and CD4+ and CD8+ SP thymocytes from C57Bl/6 and 129S6 mice. RNA levels were normalized to 18S RNA. Graphs show relative quantification calibrated to C57Bl/6 DP sample for one representative of three experiments. C. Ly108 isoform expressing EL-4 cell lines. Ly108 was immunoprecipitated from stable cell lines expressing individual isoforms using Ly108 monoclonal antibody followed by ± treatment with PNGase F. Ly108 was detected by western-blotting with pan-αLy108 (top panel) or αLy108-2 (bottom panel). Arrows indicate the predicted size of each isoform species including 8 kDa from the TAP-tag. Molecular weights including the TAP-tags are Ly108-1=44.4 kDa, Ly108-2=46.6 kDa and Ly108-H1=43.8 kDa. D. Differential expression of Ly108 isoforms in thymi from C57Bl/6 and 129S6 mice. Ly108 was immunoprecipitated from thymocytes and a portion treated with PNGase F. Ly108 isoforms were detected by immunoblotting with pan-αLy108 (top panel) and αLy108-2 (bottom panel) and are indicated by arrows. C-D are representative of 3 or more experiments.