Can anyone provide me with the protocol to do serum starvation and amino acid starvation in HeLa or HEK cells in order to measure S6K1 phosphorylation? I have a modified dmem that I use for my experiments which is similar to the commercially available dmem except contains hepes to buffer. When I do overnight serum starvation and 2 hrs amino acid starvation, I expect to see a significant decrease in S6K1 phosphorylation levels, but I get phosphorylation level similar to my controls (not deprived). Any suggestions?