Journal: Endocrine-Related Cancer
Article Title: Functional consequences of the first reported mutations of the proto-oncogene PTTG1IP/PBF
doi: 10.1530/ERC-16-0340
Figure Lengend Snippet: Expression, localisation and half-life of PBF mutations. (A) Subcellular localisation of each HA-tagged mutant (green) within HeLa cells using an anti-HA antibody. Blue indicates nuclear DAPI staining. As HA-tagged R140W is difficult to detect through immunofluorescent microscopy, FLAG-tagged wild-type (WT) and R140W PBF were stained using an anti-FLAG antibody. Magnification = 100×. Bars = 20 µm. (B) Anisomycin half-life assays in MCF7 cells transfected with HA-tagged WT PBF and 9 mutations and treated for 0, 12 or 24 h, with quantification of relative PBF levels shown in Western blotting (FLAG-tagged WT and R140W are shown alongside). (C) Quantification of protein stability of the ~25–37 kDa isoforms of PBF in MCF7 cells following normalisation to β-actin expression. Data presented as mean values ± s.e. ( n = 2). (D) SIFT scores of all ten PBF mutations, where scores <0.05 are predicted to represent deleterious amino acid substitutions, which are defined as ‘damaging.’ (E) Western analysis of lysate from COS-7 cells transfected with vector only (VO), WT PBF and the 10 PBF mutants showing the expression levels and multiple forms of the HA-tagged PBF proteins. (F) PNGase F treatment of lysate from COS-7 cells transfected with WT PBF-HA results in a band at ~20 kDa and confirms N-linked glycosylation of PBF. PBF with a double substitution of N45 and N54 for alanine is also detected at ~20 kDa and cannot be modified by PNGase F confirming these are the only sites of glycosylation. (G) Western blotting of lysate from COS-7 cells transfected with HA-tagged PBF with discrete mutation of N45 and N54 suggests that both sites are glycosylated. (H) Western analysis of WT PBF-HA expressed in HeLa cells using non-reducing (minus β-mercaptoethanol (β-ME)) conditions clearly reveals the presence of a dimer ~50 kDa. (I) Western blotting demonstrating the presence of the ~50 kDa band, and therefore the ability of FLAG-PBF to form dimers, in COS-7, HeLa and MCF7 cells transfected with WT FLAG-PBF and VO control. (J) Proximity ligation assays (PLAs) demonstrating an interaction between FLAG-PBF and PBF-HA in HeLa cells (red spots indicate <30–40 nm distance between the anti-FLAG and anti-HA antibody epitopes and signify protein–protein interaction). VO co-transfections with either FLAG-PBF or PBF-HA represent negative controls.
Article Snippet: COS-7 African green monkey kidney epithelial and HeLa human cervical carcinoma cancer cell lines were acquired from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK) and maintained in DMEM, high glucose (Sigma).
Techniques: Expressing, Mutagenesis, Staining, Microscopy, Transfection, Western Blot, Plasmid Preparation, Glycoproteomics, Modification, Control, Ligation
