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Statistical analysis Statistical analysis was performed using GraphPad Prism Software
Observation of morphological changes and counting for number of autophagic vesicles. Phase contrast morphological images of Caco-2 Deazaneplanocin were obtained after 3 days of IMQ treatment. The Caco-2 cells were seeded at 2 × 105 cells in 6-well culture plates and allowed to attach to plates for 2 days, after which IMQ was added. The cellular morphology was observed under phase-contrast microscope (Motic, Hong Kong). Autophagic vesicles were counted in 50 cells per treatment after taking representative pictures. The mean numbers of autophagosomes ± SE per cells were shown.
Western blotting analysis. Total cell lysates were prepared for Western blotting analysis. The Caco-2 cells were harvested and lysed in a buffer containing 1% SDS, 1% Triton X-100, 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 2 mM PMSF after PBS washing twice. Twenty micrograms of total extracted proteins were applied per lane for SDS–PAGE. After transfer to nitrocellulose membranes, protein expression levels were detected with specific antibodies to LC3, Ik-B, E-cadherin, puma, bcl-xl, PARP, caspase-3 (Cell signaling Technology, Danvers, MA), and α-tubulin (Sigma, St. Louis, MO).





 
 
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