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Fig. 4 | Journal of Translational Medicine

Fig. 4

From: LINC00478-derived novel cytoplasmic lncRNA LacRNA stabilizes PHB2 and suppresses breast cancer metastasis via repressing MYC targets

Fig. 4

LacRNA physically interacts with PHB2 in breast cancer cells. a Full-length sense and antisense LINC00478 with a streptavidin RNA-pulldown assay and PHB2 immunoblotting. b Immunoblotting for PHB2 with biotinylated LacRNA and LINC00478_3′RNA using streptavidin RNA-pulldown assays. c Binding of LacRNA to the PHB2 complex shown by RNA-immunoprecipitation and qRT-PCR. MEG3 was used as a negative control. d Immunoblotting of PHB2 in pulldown samples by full-length biotinylated-LacRNA or truncated biotinylated- LacRNA motifs (#1: 1–300 nt; #2: 301–600 nt; #3: 601–791 nt;), with GAPDH as the negative control. e CLIP-qPCR analysis to map interaction between LacRNA and PHB2. Following the initial PAR-CLIP steps, partial digestion aimed at generating RNA fragments ranging 100–300 nt was performed using RNase T1. After IP using PHB2 antibody and extraction of RNA, PCR amplification was used to quantify the relative abundance of overlapping segments spanning LacRNA. f Immunoblotting of PHB2 in pulldown samples by full-length biotinylated LacRNA or biotinylated LacRNA with deleted motifs (△1: 1–60 nt deleted; △2: 61–140 nt deleted; △3: 141–200 nt deleted; △4: 201–300 nt deleted). g, h Truncation domains of PHB2 that bind to LacRNA according to RNA-immunoprecipitation and qRT-PCR. Values are expressed as the mean ± SEM, in c and h., P values were determined by Student's t-test. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus IgG. IP, immunoprecipitation; IB, Immunoblotting. Uncropped images of the blots are shown in Additional file 1: Fig. S9

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