Figure 1

RNA and protein expression of TIPT2. (A) TIPT2 transcripts in mouse adult organs and embryonic (E14) tissues detected by Northern blotting. Beta actin was used as a loading control. (B) TIPT2 transcript in embryonic tissues prepared at the indicated stages of development detected by RT-PCR. a, m, and p RNAs were prepared from anterior, middle and posterior embryonic regions, respectively. (C) Bacterially made TIPT2 proteins. Lane 1: Marker. Lane 2: Coomassie blue stained recombinant GST-TIPT2. Lane 3: TIPT2, generated by thrombin cleavage, removal of GST by glutathione chromatography, and thrombin removal by benzamidine chromatography. Lane 4: Material equivalent to the protein in lane 3 was cut from a gel and electrophoresed again. (D) TIPT2 proteins detected by purified anti-TIPT antibodies in HeLa or U2OS whole cell extracts (lane 2 and 3). Detection of TIPT2 on the Western blot was competed by the inclusion of GST-TIPT2 in the antibody binding solution in a 10:1 ratio (lane 4). Markers were run on lanes 1 and 5. (E) TIPT2 proteins in organ extracts from P2 mice, adult mouse testis and a stable U2OS cell line expressing FHM-TIPT2. Asterisks indicate additional proteins recognized by anti-TIPT2 antibodies. (F) Cytoplasmic, nucleoplasmic and nucleolar localization of TIPT2 in human U2OS tissue culture cells. Nucleoli were detected with anti-TBPL1 antibodies. DNA was stained with DAPI. Bar, 20 μm. Further immunohistochemistry is presented in Additional file 1.