Journal: PLoS ONE
Article Title: Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells
Figure Lengend Snippet: Cend1 and RanBPM interact in vitro and form complexes. ( a ) In a cell-free system, a 942 bp fragment corresponding to the SPRY-LISH-CTLH domain of RanBPM was 35 S-labeled during coupled transcription/translation reactions and was then used in GST-pull down assays with GST-Cend1 protein or GST alone as negative control. GST-Cend1 specifically precipitates the SPRY-LISH-CTLH domain of RanBPM (upper panel). GST and GST-Cend1 protein loading is shown in the lower panel. ( b ) Reciprocal GST-pull down assays were performed in solubilized fractions of mouse brain homogenate where Cend1 is expressed in abundance (upper panel, input). GST-RanBPM (981bp) and GST-RanBPM (1731bp) fusion proteins precipitate the native Cend1 protein from mouse brain (middle panel). GST protein loading is shown in the lower panel. Cend1 immunoprecipitation with polyclonal anti-Cend1 antibody served as a positive control. ( c ) HEK 293T cells were transiently transfected with FLAG-tagged RanBPM and GST pull-down assays were performed with GST-Cend1 or GST as negative control (GST protein loading, upper panel). RanBPM (input, middle panel) was specifically precipitated by GST-Cend1 (lower panel). ( d ) Cend1 and RanBPM associate in vivo in HEK 293T cells. HEK293T cells were transiently transfected with Cend1, FLAG-RanBMP or both as indicated. Immunoprecipitation of RanBPM from HEK293T cell lysates was performed using anti-FLAG (upper panel) and detection of co-immunoprecipitated Cend1 was performed by immunoblotting using anti-Cend1 (lower panel). An irrelevant polyclonal antibody against peroxiredoxin II was used for immunoprecipitation, as negative control. ( e ) Cend1 and RanBPM co-localize in the cytoplasm of transiently co-transfected Neuro 2a cells. Double immunofluorescence labeling and confocal microscopy showed that Cend1 (red) is localized in the cytoplasm ( i ), while RanBPM (green) is partitioned in both the cytoplasm and the nucleus as detected by an anti-FLAG antibody (ii). Nuclei (blue) were visualized using TO-PRO-3 (iii) and the merged picture, including orthogonal optical sectioning, is shown in (iv). In (v-viii) shown is staining for the same antigens, but RanBPM is detected by an anti-RanBPM antibody. Scale bar: 10 μm. ( f ) Fractionation analysis of Neuro 2a cells transiently transfected with RanBPM followed by Western blot analysis, confirmed that RanBPM is distributed in both the cytoplasmic (C) and nuclear fractions (N). Fraction purity was checked using anti-GAPDH (cytoplasmic marker) and anti-PH3 (nuclear marker) antibodies, respectively.
Article Snippet: Other antibodies also used in this study were rabbit polyclonal antibodies against β-tubulin (sc-9104), α-actin and β-actin (sc-1615), mouse monoclonal antibody against βIII-tubulin (Tuj1; Covance, MMS-435P), mouse monoclonal antibody against green fluorescent protein (GFP) (Invitrogen), rabbit polyclonal against phospho-histone 3 (PH3) (Upstate) and goat polyclonal antibody against Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, sc-20357).
Techniques: In Vitro, Labeling, Negative Control, Immunoprecipitation, Positive Control, Transfection, In Vivo, Immunofluorescence, Confocal Microscopy, Staining, Fractionation, Western Blot, Marker