Hek293T cells were seeded on glass coverslips and allowed to adhere overnight. Levels of SIRT1 and phosphorylated protein at S47 were measured after permeabilizing with 0.1% Triton (Panel 1) and methanol (Panel 2) followed by Lambda phosphatase treatment (Panel B). The total SIRT1 signal was labeled with GAM-488 and the SIRT1 pS47 with GAR-594. Panel B shows a reduction in phosphorylation due to LP treatment.
Hek293T cells were seeded on an amine coated plate at 12k, 25k and 50k/well the day before fixation. Levels of total SIRT1 and phosphorylated protein at S47 were measured after permeabilizing with methanol at -20⁰C for 25 minutes. Once the methanol was washed with PBS, treatment with 1:100 dilution of LP was carried out at 40˚C for 45 minutes on a plate heater. Blocking and antibody incubations were carried out according to this protocol. Data is shown as the mean of different wells at different cell densities after normalization with Janus green.
Cells were seeded the day before at the specified cell densities. The signal was obtained using this kit as described. Total SIRT1 (TOP) and SIRT1 phospho S47 (BOTTOM) are shown after background subtraction.