Computational protocol: Silence of MACC1 expression by RNA interference inhibits proliferation, invasion and metastasis, and promotes apoptosis in U251 human malignant glioma cells

Similar protocols

Protocol publication

[…] The cells were harvested and lysed using radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Haimen, China) and phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology) for 30 min on ice. The protein extracts were subsequently centrifuged at 24,148 × g for 10 min at 4°C, and the protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The protein extracts (40 µg) were subsequently separated by 8%–12% SDS-PAGE prior to being transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then stained with 0.2% Ponceau S Red (Beyotime Institute of Biotechnology) to ensure equal loading of the proteins. The membranes were then blocked with 5% nonfat milk, and were incubated with antibodies targeting MACC1 (1:100; cat. no. bs-4293R; BIOSS, Beijing, China), and cyclin D1 (1:1,000; cat. no. WL0205), cyclin E (1:1,000; cat. no. WL0055), cyclin B (1:1,000; cat. no. WL0023), cleaved caspase-3 (1:200; cat. no. WL0146), B cell lymphoma 2 (Bcl-2; 1:500; cat. no. WL0104), Bcl-2-associated X protein (Bax; 1:500; cat. no. WL0101), matrix metalloproteinase 2 (MMP-2; 1:500; cat. no. WL0657), MMP-9 (1:500; cat. no. WL0884) and β-actin (1:1,000; cat. no. WL0001a; all Wanleibio Ltd., Shenyang, China) overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody at 1:5,000 dilution, at room temperature for 1 h (cat. no. A0208; Beyotime Institute of Biotechnology). β-actin was used as the internal positive control. The immunocomplexes were visualized using enhanced chemiluminescence western blotting detection reagents (7seabiotech, Shanghai, China). The relative amounts of transferred protein were quantified by scanning the autoradiographic films using UN-SCAN-IT Gel Analysis software (Silk Scientific Inc., Orem, UT, USA) prior to being normalized to the corresponding β-actin level. The quantitative analysis of the western blot was carried out using the Gel-Pro Analyzer software (Media Cybernetics, Inc., Rockville, MD, USA). […]

Pipeline specifications

Software tools UN-SCAN-IT gel, Gel-Pro Analyzer
Application DNA fingerprinting
Organisms Homo sapiens
Diseases Colonic Neoplasms, Glioma, Neoplasms