Figure 5. Themis expression.
(a) RT-PCR expression analysis using Themis exon 3 forward and exon 4 reverse primers on sorted T cell populations. Expression was calculated using the ΔΔCT method with β actin as the standard. Data are shown as the mean fold expression over B cell values for 4-12 biological replicates per population. (b) Rabbit polyclonal anti-Themis immunoblot on lysates from unfractionated homogenized tissues of a wild-type B6 and a Themis(Y489X) thymus, as well as other tissues from wild-type B6 mice. Data for thymus, LN and spleen are representative of ≥ 3 independent experiments. (c) Confocal imaging of unfixed HEK293 cells transiently transfected overnight with Themis-EGFP (green) and labeled with Hoechst 33342 nuclear dye (blue). Original magnification x63. Data are representative of 4 independent experiments. (d) Anti-Grb2 immunoblot showing specific co-immunoprecipitation with Themis-EGFP in both Jurkat and HEK293 cell lines that had been transiently transfected overnight with Themis-EGFP or EGFP control constructs. Data are representative of 3 independent experiments.