After complementary DNA was synthesized with a two-step reverse
transcription reaction kit(TAKARA, Dalian, China), quantitative PCR was performed on an Applied Biosystems 7500 Real-time PCR System using SYBR Premix Ex Taq Kit (TAKARA, Dalian, China) in Axygen 96-well reaction plates following the manufacturer’s protocols. β-actin was used as a reference to obtain the relative fold change for target samples using the comparative Ct method. SHP099 cost The primers used are as follows: β-actin forward, TCACCCACACTGTGCCCATCTACGA; β-actin reverse, CAGCGGAACCGCTCATTGCCAATGG, AQP3 forward, CACAGCCGGCATCT- TTGCTA, reverse, TGGCCAGCACACACACGATA, All cell preparations and real-time PCRs were performed in triplicate. Western blot analysis For Western blot, cells were reseeded in 6-well plates at a density of 0.2 × 106 cells/ml with fresh complete culture medium. Cells with or without treatment were washed with cold PBS and harvested by scraping into 150 μl of RIPA buffer(containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA 0.25% sodium deoxycholate) with 1mM NaF, 10 μM Na3VO4, 1 mM PMSF, and a protease inhibitor
concoction(10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 μM pepstatin). Cell lysates were incubated at 4°C for 30 min. After centrifugation at 12,000 rpm for 20 min at 4°C, protein concentrations were determined by bicinchoninic acid(BCA) protein assay. Forty micrograms of proteins(for AQP3, check details MT1-MMP, MMP-2, MMP-9, phospho-AKT or AKT) were denatured in selleck compound 5× SDS-PAGE sample buffer for 5 min at 100°C. The proteins were separated by 12% SDS-PAGE and transferred onto PVDF membrane(Millipore, Bedford, MA) for 90 min at 4°C. Nonspecific binding was blocked with 5% Mirabegron dry skimmed milk in TBST
(20 Mm Tris-HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.4) for 2 h at room temperature. After blocking, membranes were incubated with specific antibodies against AQP3(1:500), MT1-MMP(1:1,000), MMP-2(1:1,000), MMP-9(1:1,000), phospho-AKT(1:1,000), or AKT(1:1,000) in dilution buffer (2% BSA in TBS) overnight at 4°C. The blots were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:2,000) at room temperature for 2 h. Antibody binding was detected using an enhanced chemiluminescence(ECL) detection system following manufacturer’s instructions and visualized by autoradiography with Hyperfilm. Semiquantitatively analyzed of the blots were acquired using the software Quantity One(BioRad, USA). The density for AQP3, MMPs, or phospho-AKT protein in their parental sample was normalized to 1.0, and the values for other treatments were calculated against this value. Statistical analysis All data were expressed as mean ± SD. Statistical analyses were performed using Student’s t test or analysis of variance (ANOVA). The values of P < 0.05 are considered significant.