Additional file 3: Figure S3. of Coronin 2A (CRN5) expression is associated with colorectal adenoma-adenocarcinoma sequence and oncogenic signalling RastetterRaphael BlÜmacherMargit DrebberUta MarkoMarija BehrensJuliane SolgaRoxana HojeiliSarah BhattacharyaKurchi WunderlichClaudia WunderlichF. OdenthalMargarete ZiemannAnja EichingerLudwig ClemenChristoph 2015 CRN5 is methylated by PRMT5, increases cell proliferation, and sensitises cells to TRAIL-induced cell death. (A) Co-immunoprecipitation experiment using lysates from U373 cells with stable over-expression of GFP-CRN5 and anti-GFP magnetic microbeads. Precipitates were subjected to SDS-PAGE, stained with Coomassie brilliant blue, and proteins were identified by mass spectrometry analysis. Arrows indicate the positions of GFP-CRN5 and PRMT5. (B) Pull-down assay using GST-CRN5 or GST for control coupled to glutathione-coated sepharose beads and soluble His6-PRMT5 confirm a direct interaction of CRN5 and PRMT5. Proteins were expressed and purified from E. coli strain BL21. Asterisk, GST signal from partial degradation of GST-CRN5. For illustration purposes the order of lanes from the original immunoblots were digitally re-arranged to omit dispensable lanes. (C) In vitro methylation experiments based on the incubation of recombinant PRMT5/MEP50 complex, GFP-CRN5, and 3H-labelled S-adenosylmethionine showed methylation of CRN5. Controls lacked the substrate GFP-CRN5. Columns indicate mean values and standard deviations derived from three independent experiments. (D) Immunoblot verifications of PRMT5 knock-down and FLAG-CRN5 over-expression in HeLa cells. (E) Left panel: HeLa cell viability after double-transfections as indicated in absence (dark columns) and presence (light columns) of TRAIL ligand. Viability of control cells transfected with both empty vectors is set to 100 %. One representative out of ten independent experiments with similar patterns of cell viability is shown; note, that the level of absolute absorbance values varied from experiment to experiment and did not allow calculation of a mean value. Right panel: For better illustration of the results the three light columns indicate the decrease of cell viability, i.e. the rate of TRAIL-induced cell death. This was calculated by the differences of the values from the three right pairs of dark and light bars from the left panel minus the TRAIL-induced decrease of cell viability of the control cells (first left pair of bars). (TIFF 521 kb)