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. 2019 Dec 9;27(6):1844–1861. doi: 10.1038/s41418-019-0467-6

Fig. 2. Linc-ASEN associates with UPF1 and PRC1/2, and recruits UPF1-PRC1/2 complexes to the p21 gene locus.

Fig. 2

a Linc-ASEN pulldown (PD) assay was performed and the associated proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). UPF1 and PRC1/2 in Linc-ASEN RNA PD were analyzed by western blot with the indicated antibodies. b UPF1 and PRC1/2 in Linc-ASEN HA IP were analyzed by western blot with the indicated antibodies. c UPF1 associates with PRCs in a DNA/RNA-independent manner. After IP using anti-UPF1 antibody in the presence of RNase A and DNase I, the indicated proteins in precipitates were detected by western blot (upper). Actin mRNA and p21 DNA were detected to assess the extent of digestion with RNase A and DNase I (lower). d Analysis of Linc-ASEN association with UPF1 and PRCs in the nucleus and cytoplasm fractions. Lamin B and Actin were used as loading control of the nucleus and cytoplasm, respectively. e UPF1 chromatin immunoprecipitation (ChIP) was conducted in the presence or absence of Linc-ASEN. UPF1 ChIP was analyzed by western blot and RT-qPCR analyses. Enrichment of DNA was quantified relative to the amount of input. Rabbit IgG (rIgG) was used as a negative control. f ChIP analysis in the presence of UPF1 (Wt) and UPF1 R844C (Mut). After ectopic expression of UPF1 constructs in MCF7 cells, ChIP was performed using Flag antibody. Western blot and RT-qPCR analysis were performed. Enrichment of DNA was quantified relative to the amount of input. In d–f, data are represented as means ± SD. *P < 0.05, **P < 0.01 (Student’s t-test).