Research Paper Volume 12, Issue 21 pp 21129—21146

Figure 4. LINC00452 protects ROCK1 from ubiquitin-proteasome-mediated degradation. (A and B) interaction profile (A) and interaction matrix (B) showing catRAPID fragments-based prediction of interaction between LINC00452 and ROCK1. (C) catRAPID graphic output confirming the interaction of LINC00452 and ROCK1 as predicated by catRAPID fragments. (D) RIP results showing ROCK1 antibody specifically immunoprecipitated more LINC00452 from LINC00452-overexpressed CaOV3 cell lysates than control cells. Antibody against IgG was used as a negative control. M, DNA marker; LNC (OE), LINC00452 overexpression. ** p < 0.01 versus IgG group. (E) RNA pull-down assay using biotin-labeled LINC00452 yielded less ROCK1 protein when knocking down LINC00452 in CaOV3 cells. Beta-actin was used as a negative control indicating specificity of the interaction between LINC00452 and ROCK1. (F) In Truncated RNA pull-down assay, full-length LINC00452, antisense LINC00452, and truncated LINC00452 (1-190nt,1-1000nt,284-2036nt,190-2036nt) were synthesized. Results suggested that the binding region of ROCK1 protein in LINC00452 is most likely located in the 903-968 nt. (G) Western blot results showing knockdown of LINC00452 shortened the half-life of ROCK1, which could be reversed by blocking proteasome-mediated protein degradation with MG132. CHX, cycloheximide. (H) Co-immunoprecipitation of ROCK1 protein and ubiquitin. Knockdown of LINC00452 increased while overexpressing LINC00452 decreased ubiquitination of ROCK1 protein, respectively. Normal IgG was used as a negative control for IP.