Journal: Oncology letters

Article Title: Isoliquiritigenin inhibits cell proliferation and migration through the PI3K/AKT signaling pathway in A549 lung cancer cells.

doi: 10.3892/ol.2018.9344

Figure Lengend Snippet: Figure 3. ISL treatment inhibits the activation of the PI3K/AKT signaling pathway in A549 cells. (A) Effects of ISL on protein expression of AKT, mTOR, p‑AKT, p‑mTOR, P70 and Cyclin D1 in A549 cells, determined using western blot analysis. β‑tubulin was used as the loading control. (B) Quantitative results of protein expression. The data was calculated using mean ± standard deviation of three repeats. *P<0.05 compared with NC. NC, negative control; ISL, Isoliquiritigenin; PI3K, phosphatidylinositol 3‑kinase; AKT, AKT serine/threonine kinase; mTOR, mammalian target of rapamycin; p, phosphorylated.

Article Snippet: A total of 20 μg of the total proteins were fractionated electrophoretically by 10% SDS-PAGE gels (Beyotime Institute of Biotechnology, Beijing, China) and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA), which were then blocked with 5% nonfat milk for 1 h at room temperature, and then cultured overnight at 4 ̊C with the following primary antibodies: E-cadherin (dilution, 1:1,000; cat. no. 20874-1-AP; rabbit polyclonal), N-cadherin (dilution, 1:1,000; cat. no. 22018-1-AP; rabbit polyclonal), vimentin (dilution, 1:1,000; cat. no. 10366-1-AP; rabbit polyclonal), Bcl-2 (dilution, 1:1,000; cat. no. 12789-1-AP; rabbit polyclonal), Bax (dilution, 1:1,000; cat. no. 50599-2-lg; rabbit polyclonal), active caspase-3 (dilution, 1:1,000; cat. no. 19677-1-AP; rabbit polyclonal), cyclin D1 (dilution, 1:1,000; cat. no. 60186-1-lg; mouse monoclonal), P70 (dilution, 1:1,000; cat. no. 14485-1-AP; rabbit polyclonal) and β-tubulin (dilution, 1:5,000; cat. no. 10094-1-AP; rabbit polyclonal; all from ProteinTech Group, Inc. Chicago, IL, USA), AKT (dilution, 1:1,000; cat. no. 4691; rabbit polyclonal), p-AKT (dilution, 1:1,000; cat. no. 4060; rabbit polyclonal), mTOR (dilution, 1:1,000; cat. no. 2983; rabbit polyclonal) and p-mTOR (dilution, 1:1,000; cat. no. 5536; rabbit polyclonal) (all from Cell Signaling Technology, Inc., Danvers, MA, USA). β-tubulin was used as a loading control.

Techniques: Activation Assay, Expressing, Western Blot, Control, Standard Deviation, Negative Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus.

doi: 10.1038/labinvest.2011.199

Figure Lengend Snippet: Figure 2 Expression and interaction of Src-suppressed protein kinase C substrate (SSeCKS) and cyclin D1 in cultured parietal epithelial cells (PECs). (a) Fluorescence detection of SSeCKS (green), cyclin D1 (red), and nuclei (blue) during increasing cell–cell contact between PECs from 50 to 100% cell confluency showing induction of SSeCKS in the cytoplasm with concomitant nuclear to cytoplasmic redistribution of cyclin D1, compartmentalization that remains in fully differentiated (FD) PECs. (b) Western immunoblots (IB) showing downregulation of cyclin D1 and its immunoprecipitation (IP) with cytoplasmic SSeCKS during increasing cell–cell contact between PECs from 50 to 100% cell confluency, binding that remains in fully differentiated PECs.

Article Snippet: For immunoprecipitation of cyclin D1 with SSeCKS, 100 mg of protein from capsulated glomeruli or from cultured, differentiated PECs was incubated with rabbit polyclonal anti-SSeCKS antibody overnight at 41C, followed by binding to protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for SDS-PAGE separation of cyclin D1 and SSeCKS.

Techniques: Expressing, Cell Culture, Fluorescence, Western Blot, Immunoprecipitation, Binding Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus.

doi: 10.1038/labinvest.2011.199

Figure Lengend Snippet: Figure 3 Phosphorylation of Src-suppressed protein kinase C substrate (SSeCKS) by activated protein kinase C (aPKC) in cultured parietal epithelial cells (PECs). (a) Western immunoblots showing rapid phosphorylation of SSeCKS at serine 283 (phospho-SSeCKS ser283) within 5 min of activation of PKC in PECs by phorbol 12,13-diacetate (PDA) or tumor necrosis factor-a (TNF-a), signaling abrogated by staurosporine aglycone (inhibitor). (b) Control western immunoblots showing the same signaling as in panel a in mouse fibroblasts expressing SSeCKS. (c) Fluorescence detection of phospho-SSeCKS ser283 (green), cyclin D1 (red), and nuclei (blue) in fully differentiated PECs showing phosphorylation of SSeCKs and cytoplasmic-to-nuclear redistribution of cyclin D1 following activation of PKC.

Article Snippet: For immunoprecipitation of cyclin D1 with SSeCKS, 100 mg of protein from capsulated glomeruli or from cultured, differentiated PECs was incubated with rabbit polyclonal anti-SSeCKS antibody overnight at 41C, followed by binding to protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for SDS-PAGE separation of cyclin D1 and SSeCKS.

Techniques: Phospho-proteomics, Cell Culture, Western Blot, Activation Assay, Control, Expressing, Fluorescence