I don't see much difference in 2-NBDG uptake with or without phlorizin? I use to incubate HEK293 cells with phlorizin for 10-15 min. Should I leave it longer before adding 2-NBDG?
Preincubation time shouldn't make much difference as phlorizin is a competitive inhibitor of glucose for cell uptake(ie inhibition is due to the binding step which occurs at sub-second scale). I think your problem rather lies at the concentration level. What are the phlorizin and 2-NBDG concentration in your uptake assay and what is the apparent Ki of phlorizin for glucose uptake in HEK293?