Fig 1: Paupar directly binds the KAP1 chromatin regulatory protein in mouse N2A neuroblastoma cells AOverview of the pulldown assay. In vitro‐transcribed biotinylated Paupar RNA was immobilised on streptavidin beads and incubated with N2A cell nuclear extract. Bound RNA protein complexes were extensively washed and specific Paupar‐associated proteins, which do not interact with a control mRNA of a similar size, identified by mass spectrometry.BGene Ontology terms were used to annotate Paupar‐associated proteins according to biological process. The Bonferroni correction was used to adjust the P‐values to correct for multiple testing.CEndogenous Paupar transcript interacts with transcription and chromatin regulatory proteins in N2A cells. Paupar association with the indicated proteins was measured using native RNA‐IP. Whole cell lysates were prepared and the indicated regulatory proteins immuno‐precipitated using specific antibodies. Bound RNAs were purified and the levels of Paupar and the U1snRNA control detected in each RIP using qRT–PCR. Paupar transcript directly interacts with KAP1 and RCOR3 in N2A cells.D, ENuclear extracts were prepared from UV cross‐linked (D) and untreated (E) cells and immuno‐precipitated using either anti‐KAP1, anti‐RCOR3 or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and the U1snRNA control transcript were detected in each UV‐RIP using qRT–PCR.FPAX6 associates with KAP1 in N2A cells. FLAG‐PAX6 and KAP1 or RCOR3 expression vectors were transfected into N2A cells. Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. Co‐precipitated proteins were detected by Western blotting.Data information: For RNA‐IP and UV‐RIP assays, results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3. One‐tailed t‐test, unequal variance *P < 0.05, **P < 0.01, ***P < 0.001.Source data are available online for this figure.