Fig. 2
A small peptide (EPRp) originates from EPR and displays junctional localization. a Schematic of the exon−intron structure (not in scale) of EPR, a putative ORF is dashed. Percentage of human/mouse identity in the putative ORF and in its flanking RNA regions is presented. b Alignment of the predicted mammalian amino acids sequence encoded by the putative ORF present in EPR and in its orthologs. The position of the predicted transmembrane α-helix is shown as a solid black bar while the position of an additional cytoplasmic α-helix is shown as a solid green bar. c Left, diagram of the FLAG-fusion construct used for transfection (EPRp-FLAG); right, immunoblot analysis of total cell extracts from either mock or EPRp-FLAG-transfected HEK-293 cells. d Immunoblot analysis of total cell extracts from NMuMG cells serum-starved and either treated with TGF-β (+) for 24 h or untreated (−); extracts from EPRp-FLAG-transfected HEK-293 cells (EPRp-FLAG) represent a positive control. e SDS-PAGE analysis of total cell extracts from either mock or EPRp-FLAG-overexpressing (EPRp-FLAG) NMuMG cells immuno-purified using anti-FLAG monoclonal antibody. A representative silver-stained gel is shown. Asterisks indicate the position of immunoglobulin heavy and light chains. f Coimmunoprecipitation of FLAG-tagged EPRp and distinct junctional proteins (as indicated) in total extracts from NMuMG cells stably transfected with EPRp-FLAG. g Immunofluorescence analysis of either mock or EPRp-FLAG- or EPRpSTOPE-FLAG-stably transfected NMuMG cells cultured to confluence, to allow formation of cell−cell junctions. Arrows point to CGN and EPRp-FLAG junctional localization. Scale bar: 10 μm. For immunoblots, the indicated antibodies were used; the position of molecular mass markers is presented on the left and representative gels are shown. ACTB is also known as Actin Beta
