Fig. 5

RPS7 regulates LOXL2 expression via binding to LOXL2 mRNA. A RNA immunoprecipitation assay was performed in MHCC97H cells by using antibody targeting RPS7, qRT-PCR was performed to detect the mRNA level of LOXL2 in the precipitated RNA-Protein complex. B-E To further identify the specific binding region of RPS7 on LOXL2 mRNA, a series of RNA pull-down analyses were performed using different biotinylated fragments of LOXL2 mRNA including 5’UTR, CDS, 3’UTR (B), and 2576-2975 nt, 2976-3375 nt, 3376-3721 nt (C), 2976-3175 nt, 3155-3375 nt (D) within 3’UTR, as well as truncated recombinants of 3190-3259 nt (E). Western blot assay was subsequently used to detect RPS7 in these indicated mRNA fragments pull-down complex. Input showed 1% lysate. UN represented a control using beads only. NC represented a negative control using non-blot RNA. F. The pmirGLO-derived luciferase reporter containing wild-type and three different mutants of AUUUA motifs were constructed, 293T cells were transfected with these plasmids respectively. Luciferase activities normalized against Renilla luciferase activities were measured to determine the binding efficient of the AUUUA motifs to RPS7 in response to RPS7 overexpression or RPS7 knockdown. *, P < 0.05, **, P < 0.01, ***, P < 0.001. ns, no significant