D 1.Relative miRNA level1 0.8 0.6 0.4 0.2miR-96 miR-182 miR-EV GSKRela ve amount of nuclear -Catenin3 2 1 0 WT KOFigure 2. KO of GSK3b increases protein level and nuclear translocation of b-Catenin. (A) GSK3b KO improved b-Catenin expression level. Wholecell lysates have been prepared from WT or GSK3b KO MEF cells, respectively, and protein levels of GSK3b, b-Catenin, CK1e, CK2a and b-Actin were resolved by western blotting (WB). (B) b-Catenin protein translocates into the nucleus in GSK3b KO MEF cells. Cytoplasmic and nuclear fractions were prepared from WT or KO MEF cells, respectively, and b-Catenin protein levels were determined by WB. (C) MiR array analysis showed that GSK3b KO enhanced the expression of miR-96, miR-182 and miR-183 6-, 5- or 3-fold, respectively. (*P 0.05, by Student’s t-test). (D) Enhance of GSK3b protein level inhibited the expression of miR-96, miR-182 and miR-183 in AGS cells. A construct encoding GSK3b was transfected into AGS cells. Forty-eight hours immediately after transfection, total RNA was extracted and used for RT-PCR.Tween 20 site All experiments have been repeated 3 times with related outcomes (*P 0.Oxfendazole supplier 05 by Student’s t-test).Nucleic Acids Investigation, 2014, Vol. 42, No. 5ARela ve GSK3 protein levels 1.4 1.2 1 0.eight 0.6 0.four 0.2 0 1 Rela ve GSK3 protein level 1.2 1 0.eight 0.six 0.4 0.two 0 Typical(N) Tumor(T) 2 three four five six 7Normal TumorBRela ve -Catenin protein levels 6 5 4 3 2 1 0 1 Rela ve -Cateninprotein level five four 3 two 1 0 Regular(N) Tumor(T) 2 three 4 five six 7Normal TumorC three.Rela ve mature miRNA level three two.5 2 1.five 1 0.5Normal TumorRela ve pri-miR-183 levelD three.three two.five two 1.5 1 0.5 0 NormalmiR-miR-miR-TumorFigure 3. Expression levels of GSK3b, b-Catenin, miR-96, miR-182, miR-183 and pri-miR-183 in human gastric cancer. (A) GSK3b protein levels in eight human gastric cancer tissues and matched regular tissues determined by WB. The integrated intensity (counts-mm2) of each and every GSK3b band was quantified and normalized with that of respective GAPDH. The upper panel shows individual quantifications. Statistical evaluation of the normalized density is shown in bottom panel. GSK3b protein level decreased 2-fold in gastric cancer (n = 8, *P 0.05 by Student’s t-test). (B) b-Catenin protein levels in eight human gastric cancer tissues and matched typical tissues determined by WB.PMID:23509865 The integrated intensity (counts-mm2) of every b-Catenin band was normalized with that of respective GAPDH. The upper panel shows person quantifications. Statistical evaluation with the normalized density is shown in bottom panel. b-Catenin protein level elevated 3-fold in gastric cancer (n = 8, *P 0.05 by Student’s t-test). (C) The expression levels of miR-96, miR-182 and miR-183 had been enhanced in gastric cancer samples compared together with the matched typical tissues. Total RNA was extracted employing TRIZOL and miRs have been measured by signifies of TaqMan real-time RT-PCR miR detection kits. (D) The pri-miR-183 level in gastric cancer samples and inside the matched typical tissues. Total RNA in the tumor and matched regular tissues was used for RT-PCR to measure pri-miR-183 level. All RT-PCR experiments have been performed in triplicate (n = eight, *P 0.05 by Student’s t-test).KO of GSK3b increases protein level and nuclear translocation of b-Catenin GSK3b phosphorylates b-Catenin which is primed by other kinases such as casein kinases 1 and 2, a needed prerequisite to its entry into the ubiquitin-proteasome pathway for degradation (5). We initially quantified protein levels of b-Catenin, GSK3b, CK1e and CK2a in WT and GSK3b KO.
