Cell cultures were treated with 10 nM recombinant human chemerin21 157 at various time points. Cultures added medium only served as controls, and a MEK12 inhibitor U0126 was added to some cultures one hour prior to challenge with 10 nM chemerin. A number of 0. 5 x106 cells were seeded per well in a six multiwell plate and grown in a culture medium with 10% FCS for 24 h. Subsequently, the cells were washed twice in PBS and grown under reduced serum conditions for 24 h. Thereafter, cultures were washed twice and challenged with 10 nM chemerin for 1 minute, 2. 5 minutes, 5 min utes and 10 minutes. Cells were then harvested directly in 150 ul SDS buffer containing NuPAGE LDS sample buffer, NuPAGE Redu cing agent, phosphatase inhibitor, protease inhibitor, and distilled water.
The amount of total protein was measured in each lysate using Modular E 170. The samples were heated to 100 C for five minutes before an equal amount of protein from each extract were loaded into different wells. A total of 15 ul of a pre stained protein marker was added to control selleck chemicals the efficacy of the electrophoresis. Ten ul of a biotinylated protein ladder to assess the molecular weights of proteins were also added. Proteins were sepa rated by electrophoresis in NuPAGE Mes SDS running buffer at 200 V, using 100 125 mA per gel for 35 min. Electroblotting was performed by electrontransfer onto PVDF membranes in NuPAGE transfer buffer with 10% methanol at 30 V, using 170 mA per gel transfer for 1 h. After electroblotting, the mem branes were blocked with 5% non fat dry milk0. 1% Tween 20 for 1 h at room temperature.
Next, the membranes were incubated with primary antibodies overnight at 4 C in 5% BSA0. 1% Tween 20. The phospho p4442 antibody was used at a 12000 dilution and the phospho Akt anti body was diluted at 11000. To control for equal load ing amounts the membranes were incubated with b actin antibody, dilution 11000. The membranes were then washed and a knockout post incubated with horseradish peroxidase conjugated goat anti rabbit IgG and HRP conjugated anti biotin antibody for 1 h at room temperature. Blots were detected by adding substrate containing Lumiglo reagent and peroxide and developed with Fujifilm LAS 3000. A densito metric comparison between the protein bands was per formed using the GeneTools software. Cytokine and metalloprotease measurements Chondrocyte cultures were incu bated for 24 h in medium supplemented with 10% serum. Then, the cells were washed twice with PBS and further incubated for 24 h and grown under reduced serum conditions. The cultures were then washed twice and one culture received medium with 10 nM chemerin21 157, another received medium with 100 nM chemerin21 157 and a third was added a medium with vehicle only as control.

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