The solution was poured off and the cells incu bated sequentially

The solution was poured off and the cells incu bated sequentially with anti COX 2 epitope specific antibody or anti phos pho ERK12 for 60 minutes, bioti nylated secondary antibody for 45 minutes, and horseradish peroxidase conjugated streptavidin for 20 minutes. Between each incubation cells were washed with Tris buffered salineTween three times. The chro mogen 3 amino 9 ethylcarbazole was then added for 15 minutes and finally counterstained with Mayers hematoxy lin. The cells were mounted with a coverslip and visualized under light microscopy. Reverse transcription PCR Total RNA was isolated from cells after a 12 h co culture of synovial fibroblasts and C. albicans using easy BLUE Total RNA Extraction Kit. For first strand cDNA synthesis, 3g of total RNA was used in a single round RT reaction, con taining 0.
75g oligo 14 primer, 1 mM deoxynucleosides, 1first selleck chemical OAC1 strand buffer, 0. 4 mM dithiothreitol, 40 units RNaseOut recombinant ribonuclease inhibitor, and 200 units of superscript II reverse transcriptase. The reverse transcription reaction was performed at 42 C for 2 h, followed by 95 C for 5 minutes. PCR was run using 0. 9l of the reverse transcription reaction mixture as template, 0. 4 mM of gene specific primers, 1PCR buffer, 0. 25 mM dNTPs, and 1. 5 units of Taq DNA polymerase. The amplification was carried out at 94 C for 1 minute, then for 30 cycles at 94 C for 1 minute, 56 C for 1 minute, and 72 C for 1 minute followed by a final extension at 72 C for 10 minutes. All PCR products were size fractionated by a 1. 5% agarose gel electrophoresis, and DNA bands were visualized by staining the gel with 0.
1g ml ethidium bromide. The bands were analyzed using gel inhibitor MEK Inhibitor documentation system. The values were expressed as ratio of the band intensity of the target gene to glyceraldehyde 3 phosphate dehydrogenase and the ratio of the band intensity of COX 2 GAPDH in the control condition was normalized to 1. Variance and P values were analyzed by Alphaimager 1220 V5. 5. A Student t test was used for statistical comparison between groups. A P value of less than 0. 05 was considered statistically significant. Analysis of COX 2, ERK12 and phospho ERK12 expression Following C. albicans infection cells for 12 h were immediately washed with ice cold PBS containing 100M Na3VO4 and lysed in situ with ice cold lysis buffer at 4 C for 15 minutes. Lysis buffer contained 1% Igepal, 100M Na3VO4, and a protease inhibitor cocktail tablet. Whole cell lysates were collected after centrifugation at 14,500 rpm for 15 minutes. Protein concentration was determined by the Lowry method. Equal amounts of protein were loaded onto 10% SDS polyacrylamide gels and were transferred to polyvinylidene dif luoride membranes.

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