Extended Data Fig. 2: The specificity of alncRNA.
From: Engineering artificial non-coding RNAs for targeted protein degradation
(A) RIP analysis of the interaction of alncRNA with a panel of RBPs in cell lysates. Antibodies recognizing the RBPs shown were used for IP in each case; control IP reactions were carried out using a corresponding IgG. AlncRNA levels were measured by RT-qPCR and normalized to the levels of GAPDH mRNA levels in the same IP samples measured by RT-qPCR analysis. Data were quantified as enrichment of alncRNA in the RBP IP relative to the IgG IP. Data represent mean ± s.d. from three independent experiments. The P values were determined by a two-tailed unpaired Student’s t test. (B-C) The protein levels of RBPs were analyzed in alncRNA-expressing cells by WB. Data represent mean ± s.d. from three independent experiments. The P values were determined by a two-tailed unpaired Student’s t test. (D) Proteomics analysis ensures the specificity of alncRNA. The data were analyzed using a two-tailed unpaired Student’s t-test. Adjustments were made for multiple comparisons using the Bonferroni correction method. (E-F) RIP assays show that alncRNA is pulled down by Dzip3 and GFP antibody in cells. Immunoprecipitation with control IgG served as the negative control. Data represent mean ± s.d. from three independent experiments. The P values were determined by a two-tailed unpaired Student’s t test. (G) RNA pull-down assays showed that Dzip3 and GFP was pulled down by alncRNA. Antisense of alncRNA was used as negative control. (H-I) The effect of reporter gene activation was determined by dual-luciferase reporter assay. CRISPR plasmids were transfected into cells stably expressing a dual-luciferase reporter vector. Relative luciferase activities were determined as the ratios between Rluc and Fluc values. Data represent mean ± s.d. from four independent experiments. The P values were determined by a two-tailed unpaired Student’s t test. (J-K) Fold change in abundance of whole-cell proteins detected using quantitative proteomics analysis after c-Myc (J) and NF-κB (K) degradation. The data were analyzed using a two-tailed unpaired Student’s t-test. Adjustments were made for multiple comparisons using the Bonferroni correction method.
