Journal: Neoplasia (New York, N.Y.)

Article Title: The FUS-DDIT3 Interactome in Myxoid Liposarcoma 1

doi: 10.1016/j.neo.2019.05.004

Figure Lengend Snippet: Reciprocal co-immunoprecipitation and proximity ligation assays validate FUS-DDIT3’s association with NONO, PSPC1 and SFPQ. (A) Whole cell lysates from two myxoid liposarcoma cell lines, 402-91 and 1765-92, each harboring a different variant of the FUS-DDIT3 fusion oncoprotein, were used to test for reciprocal co-immunoprecipitation of the Drosophila behavior/human splicing proteins NONO, PSPC1, and SFPQ with the FUS-DDIT3 oncoprotein. For color blots, FUS was detected in the green channel and DDIT3 in the red channel, and the FUS-DDIT3 band is indicated by an overlap of FUS and DDIT3 signal to form a merged yellow-colored band. Larger images of the same blots with visible IgG bands from each IP antibody are in Figure S4 to visualize the amount of antibody used. (B) Proximity ligation assay, a technique that amplifies a red signal when two proteins colocalize within 40 nm of each other in situ , was performed in myxoid liposarcoma cell lines 402-91, 1765-92, DL-221, and negative control cell line HeLa. Red signals indicate proximity between proteins of interest. Nuclei were counter-stained with DAPI (blue). Scale bar = 5 μm.

Article Snippet: Antibodies used for immunoprecipitations were: normal mouse or rabbit IgG (Santa Cruz), DDIT3 (L63F7) (Cell Signaling Technology, #2895), NONO (Abcam, ab70335), PSPC1 (Abcam, ab104238), and SFPQ (Novus Biologicals, NB-100-61045).

Techniques: Immunoprecipitation, Ligation, Variant Assay, Proximity Ligation Assay, In Situ, Negative Control, Staining