Journal: Oncology Letters

Article Title: PRL-3 promotes cell adhesion by interacting with JAM2 in colon cancer

doi: 10.3892/ol.2016.4836

Figure Lengend Snippet: PRL3 interacts with JAM2. (A) The candidate interaction protein of PRL-3 in a yeast two hybrid system. (B) Interaction between PRL-3 and JAM2. Cells were cotransfected with PEBG-JAM2, PcDNA3-myc-PRL-3 plasmids and its control. Protein lysates were immunoprecipitated with an anti-myc antibody or GST antibody. Precipitates and protein were analyzed by western blotting with antibodies against GST and myc. (C) Colocalization of PDSRED-JAM2 and GFP-PRL-3 in LoVo cells. Cells were cotransfected with PDSRED-JAM2 and GFP-PRL-3 vector for 48 h. Scale bar, 25 µM. JAM2, junctional adhesion molecular 2; IgG H, IgG heavy chain.

Article Snippet: Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes (Hybond-C; #RPN303C; GE Healthcare Life Sciences, Little Chalfont, UK), which were then blocked in 5% skim milk in PBS-Tween, and probed with the indicated antibodies [mouse monoclonal anti-myc (#TA100010) or anti-GST (#TA150102) antibodies (OriGene Technologies, Inc.); final concentration 1 µg/ml) at 4°C overnight, washed with 0.1% Tween-PBS three times, then incubated with horseradish peroxidase-linked horse anti-mouse IgG antibody (#7076; Cell Signaling Technology, Inc., Danvers, MA, USA; dilution, 1:2,000) at room temperature for 45 min).

Techniques: Immunoprecipitation, Western Blot, Plasmid Preparation