The proteins were subjected to electrophoresis and transferred onto nitrocellulose membranes

ization was then carried out to remove embedded medium using xylene incubation for 20-minute. TMAs were gradually rehydrated in serial alcohol baths followed by a distilled water wash for 5 min. Heat purchase Danoprevir induced epitope retrieval with trilogy was then performed to unmask PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657123 the antigenic sites within the tissue sections. TMAs were blocked in TBS containing 1% fetal calf serum and 1% bovine serum albumin for 15 min. Perox-free blocking reagent was also added for 10 min to block non-specific antibody binding. TMAs were incubated with IGF1R antibody overnight at 4uC. Slides were then washed three times in PBS for 5 min and incubated with Ultra Marque polyscan HRP Label for 1 h at room temperature. TMAs were then washed three times in PBS and stained with chromogen solution for 20 min. Chromogen staining reaction was stopped by rinsing with distilled water. Cell nuclei counterstaining was performed with hematoxylin incubation for 40 sec. TMAs were rinsed with distilled water and dehydrated with serial ethanol baths followed by a xylene bath. Finally, TMAs were coverslipped with the mounting media and digital images were captured using a Nikon Microscope- ECLIPSE 50i. Materials and Methods Ethics Statement All the experiments performed were approved by and performed following the guidelines of the Institutional Biosafety Committee of Texas Tech University Health Sciences Center. Cell Lines and Reagents Human pancreatic ductal adenocarcinoma cell lines, PANC-1, MIA PaCa-2 and HPAC were purchased from the American Type Culture Collection, and were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 Units/mL of penicillin, and 100 mg/mL of streptomycin. Cells were maintained at 37uC in a humidified atmosphere containing 5% carbon dioxide. TransIT- siQUEST transfection reagent was purchased from Mirus Bio. The 6.5 mm Transwell with 8.0 mm pore polycarbonate membrane inserts was obtained from Corning Incorporated. BD Matrigel and BD Pharmingen Annexin V-FITC Apoptosis Detection Kit I was obtained from BD Biosciences. BSA was purchased from Sigma-Aldrich Corporation. siRNA targeting IGF-1R was purchased from Origene. MTS reagent was obtained from Promega. Mammalian protein extraction reagent was purchased from Thermo Scientific. The following primary antibodies were used in this study: pAKT, AKT, Bcl-2, pERK,, ERK and STAT3; IGF-1R, Notch 2, Snail, E-cadherin, N-cadherin, Zeb, Vimentin, Slug, Bax, Caspase3, PARP, pPI3K p85, PI3K p85, IR-b, pIRS-1, IRS-1, pSTAT3 Silencing of IGF-1R in PANC-1 and HPAC Cells siRNA targeting IGF-1R were transiently transfected into PANC-1 and HPAC cells using MIrus bio TransIT siQUEST transfection reagent. Scrambled siRNA was used as a control. Briefly, cells were seeded in 6-well plates at a density of 2.56105 cells/well. Cells were transfected with different concentrations and different subtypes of siRNA ranging from 10 to 50 nM for 48 h or 72 h, using Mirus siQUEST Transfection Reagent. The ratio of siRNA to Transfection reagent was maintained as 1:0.5 for efficient silencing without toxicity according to the manufacturer’s protocol. The final concentrations of siRNA were chosen based on dose response studies. Forty-eight hours after the transfection, cells were used for protein isolation or clonogenicity, invasion, and migration, studies. Apoptosis was studied at 48 and 72 hours after silencing of IGF-1R. Role of IGF-IR in Pancreatic Cancer Cell Viability Assay PANC-1 and HPAC cells were seeded in 96-well plate

评论已关闭。