Briefly, archival tissues from canine melanomas were http://www.selleckchem.com/products/Y-27632.html identified and 5m serial sections from paraffin embed ded blocks were mounted onto positively charged slides. Immunos taining was performed using a modified streptavidin biotin complex method. Antigen retrieval was done by microwave heating for 6 minutes in a buffer of 0. 1 M sodium citrate. Samples were graded for intensity and percentage of positive cells, as well as for subcellular location of the relevant stain. Samples were considered negative when no staining was seen above that seen in negative controls. Staining that was fine, diffuse, and not always visible at low magnification, but was readily detectable in 30% of cells at high magnification was considered weak. Staining that was promi nent enough to be seen at low magnification in 30% of cells was considered moderate to strong.
Samples were only considered Inhibitors,Modulators,Libraries positive when expression of these proteins was clearly detectable in melanocytic tumor cells based on the expression of S100a protein, Melan A, or neuron specific enolase in serial sections from the tumors. Protein stability assays Approximately 2. 5 105 cells were grown on 6 well tissue culture dishes in media containing 10% fetal bovine serum. Cells were treated with 20g/ml cycloheximide at 30 min interval up to a maximum of 2. 5 h. Cells were collected and analyzed by immunoblot ting. Immunofluorescence microscopy Inhibitors,Modulators,Libraries Cells were grown on Lab Tek multi chamber slides and fixed in cold methanol for 10 min at 20 C. Fixed cells were rehydrated with PBS and blocked with 10% goat serum for 1 h at room tem perature.
Cells were incubated with anti p21 antibody for Inhibitors,Modulators,Libraries 30 min at room temperature, followed by a sec ondary goat anti mouse IgG antibody conju gated to Alexa Fluor 594 for Inhibitors,Modulators,Libraries 30 min at room temperature. DAPI was Inhibitors,Modulators,Libraries used for nuclear counterstaining. The LSM 5 Pascal confocal laser scanning microscope was used for imaging. Caspase assay The CaspACE assay kit was used to measure caspase 3 activity per the manufacturers instructions. Briefly, 106 cells per 10 cm dish were treated as described in the text. Positive control cells were treated with 4M camptothecin. The cells were harvested, washed, and equal protein quantities were incubated with the DEVD pNA caspace 3 substrate in Caspase Assay Buffer. The plates were covered with parafilm and incu bated at 37 C overnight.
Color development was meas ured at 405 nm. Subcellular fractionation Cells were grown in 100 cm dishes to 95% confluency fol lowed by separation into sellckchem nuclear and cytosolic fractions using the Panomics Nucelar extraction kit according to the manufacturers instructions. Briefly, cells were washed with PBS twice and lysed in a buffer containing 100 mM HEPES, pH 7. 9, 100 mM KCl, and 100 mM EDTA on ice for 10 minutes. Lysates were centrifuged at 15,000 g for 3 min at 4 C to obtain cytosolic extracts. Soluble nuclear extracts were obtained by vortexing nuclear pellets in a buffer containing 100 mM HEPES, pH 7.