bSMase affects backbone fluorophores FRET-independently. HeLa cells were incubated with TFSM 1 in the presence of 1% FBS. The molecule was removed, and cells were bSMase-treated, fixed, and stained with Cy5-azide and AlexaFluor TM (AF)488. Fluorescence intensities in Cy5 and AF488 channels were measured, and Cy5/AF488 ratios were calculated. Scale bars: 50 m. n = 3.
