Hi, I am studying the effect of different mutations of a protein on FGFR degradation. Here is the layout of my experiment:

HEK cells were cultured to appropriate confluency and then plated in wells. The following morning they were transfected with DNA (FGFR combined with either empty vector, or my mutations). The next day, media was removed and cells were serum starved for 2 hours, treated with 20 μg/ml cycloheximide for 1 hour and then stimulated with 100 ng/mL FGF for time periods of 0, .25, 1 and 3 hours, after which they were lysed. Samples were analyzed using SDS PAGE-western blotting. Blots were incubated with primary anti-v5 and anti-actin antibodies and secondary anti-mouse 680 antibody. 

I was originally using half the concentrations of FGF and cyclohexamide, and trials were showing increase in FGFR, not degradation, which doesn't make sense. I tried doubling the concentration of each, and it worked...for two trials. The next trials showed increases in FGFR again in the first 15 minutes with eventual degradation after 3 hours. If anyone has suggestions or ideas about what might be happening, it would be much appreciated. I started with a fresh batch of cyclohexamide, so it should be working, and I shouldn't see increasing protein levels. 

Thanks,

Rayna