Fig 1: LINC00930 interacts with RBBP5 and GCN5. a The LINC00930-interacting proteins were annotated with their Log10 ratio in 5-8F and CNE2 cell lysate. b Western blot of the proteins from antisense LINC00930 and LINC00930 pull-down assays. c RNA immunoprecipitation experiments were performed using anti-RBBP5 or anti-GCN5 antibody, and specific primers were used to detect LINC00930 or GAPDH. d Nuclear lysates of 5-8F were immunoprecipitated with anti-RBBP5 antibodies (Left), anti-GCN5 antibody (Right), or control IgG. Aliquots of nuclear lysates (20% of input) and the RBBP5 or GCN5 immunoprecipitates were separated by SDS-PAGE, and the specific immunoprecipitation of RBBP5 and GCN5 was confirmed by western blot (WB). The complexes were analyzed for the presence of LINC00930 or GAPDH by real-time PCR. e Co-IP assay detected the interaction of RBBP5 and GCN5 in the 5-8F cell. The 20% of input (cell lysate) and RBBP5 or GCN5 immunoprecipitates were separated by SDS-PAGE. The specific immunoprecipitation of RBBP5 and GCN5 was confirmed by Western blot. f IP assay was performed to detect the interaction between RBBP5 and GCN5 after LINC00930 knockdown. g Western blot of RBBP5 and GCN5 in samples pulled down by full-length (FL) or truncated LINC00930 (?1: 1–450, ?2: 451–900, ?3: 901–1350, ?4: 1351–1762). h Top: Mutation on W279, K281 and G284 of RBBP5 impaired the RBBP5-LINC00930 association. Bottom: Mutation on N291, R294 and C297 of GCN5 impaired the GCN5-LINC00930 association
Fig 2: LINC00930 functions as a link between RBBP5/GCN5 and PFKFB3. a Western blot assay was performed to measure PFKFB3 level after transfection of LINC00930-overexpressing plasmid, RBBP5 or GCN5 siRNAs in CNE1 cells. b Luciferase reporter vector was generated by inserting the promoter region (- 2000 bp to + 200 bp) of the PFKFB3 gene. The reporter vectors were then cotransfected into CNE2 cells with LINC00930 shRNA, RBBP5 or GCN5 siRNAs. Cells were harvested for luciferase activity assay. c ChIP-qPCR was performed to evaluate the enrichment of H3K4me3 or H3K9ac in different promoter regions of PFKFB3 in CNE2 cells after knocking down LINC00930. d Real-time PCR of the ChIP samples was applied to detect the binding efficiency of RBBP5 or GCN5 to the PFKFB3 gene promoter (- 800 bp to - 600 bp) after transfection of RBBP5, GCN5, or LINC00930 siRNAs in CNE2 cells, respectively. e Real-time PCR of the ChIP samples was utilized to measure the H3K4 trimethylation or H3K9 acetylation and H3K27 trimethylation levels of the PFKFB3 gene promoter (- 800 bp to - 600 bp) after knockdown of RBBP5, GCN5, or LINC00930 in CNE2 cells, respectively. f ChIRP assays of the enrichment of LINC00930 and PFKFB3 promoter (- 800 bp to - 600 bp) in both even and odd probes pools relative to control LacZ probes set in 5-8F and CNE2 cells. Data in b, c, d, e & f are presented as mean ± SEM of three independent experiments. The p-value was determined by a two-tailed unpaired Student’s t test. * p < 0.05; ** p < 0.01
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