Figure 2: CPEB1 directly interacts with the 3′UTR of p27kip1. | Scientific Reports

Figure 2: CPEB1 directly interacts with the 3′UTR of p27kip1.

From: CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27Kip1 mRNA translation

Figure 2

(A) T98G and A172 cells were transfected with the empty vector (C) or pCPEB1. After 48 h, the whole-cell lysates were run on the same gel, blotted onto nitrocellulose and the membrane cut to allow probing with antibodies against p27 or Gapdh. Relative signal intensities (right panel) for p27 in the Western blots normalized to the Gapdh loading control, are included for comparison of p27 levels. Bars represent average +/− SD of three independent experiments. Student’s t-test was utilized to calculate p values. (B) Cell lysates from T98G cells trasfected with the empty vector (C), pCPEB1 or pEZH2, were subjected to RIP assay with Flag antibody. P27 and GAPDH mRNA levels were detected using RT-PCR as shown in the representative cropped gel. (C) Proliferation assay of HeLa cells co-transfected with pCPEB1, siRNAs against p27 and relative controls was performed as in Fig. 1C. ANOVA test in combination with Dunnett’s test were performed to calculate p values, comparing the mean of each column with the mean of the control column. (D) pUTRp27 luciferase and a renilla control luciferase constructs were co-transfected with the empty vector (C) or pCPEB1. After 48 h from transfection, T98G and HeLa cells were harvested and assayed with Dual Luciferase Assay (Promega) according to the manufacturer’s instructions. Relative luciferase activity is the ratio between firefly luciferase and renilla control luciferase. Student’s t-test was utilized to calculate p values. (E) pUTRp27 luciferase constructs were co-transfected with siRNA negative control (siC) or siRNA against CPEB1, and cells were treated as in (D). (F) HeLa cells were transfected with the indicated luciferase constructs in the presence of empty vector (C) or pCPEB1 and processed as in (D). (G) Protein complexes extracted from HeLa cells, transfected with the indicated luciferase constructs, were immunoprecipitated and treated as indicated in (B). The cDNA was amplified by specific oligos corresponding to the Firefly luciferase mRNA.

Back to article page