Anti tfeb

GES-1 cells were treated with 5 μM PPIs (rabeprazole, pantoprazole, esomeprazole, or dexlansoprazole) for 1 week and then subjected to nuclear fractionation. Briefly, 5 × 10 6 cells were collected in PBS by centrifugation and washed twice with cold PBS. Subsequently, the cells were gently resuspended in 500 μL of a hypotonic buffer (20 mM Tris HCl [pH 7.4], 10 mM NaCl, and 3 mM MgCl 2 ) by pipetting up and down several times and incubated on ice for 15 minutes. Next, 25 μL of 10% NP-40 was added, followed by vortexing for 10 seconds.
The homogenate was then centrifuged for 10 minutes at 3000 rpm at 4°C. The supernatant obtained contained the cytoplasmic fraction, and the pellet obtained was the nuclear fraction. Thereafter, the nuclear fraction (pellet) was resuspended in 50 μL of a nuclear extraction buffer (10 mM Tris [pH 7.4], 2 mM Na 3 VO 4 , 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM EGTA, 0.1% sodium dodecyl sulfate, 1 mM NaF, 0.5% deoxycholate, and 20 mM Na 4 P 2 O 7 ) for 30 minutes on ice along with vortexing at 10-minute intervals. Subsequently, the sample was centrifuged for 30 minutes at 14,000g at 4°C. Finally, the supernatant (nuclear fraction) was collected and subjected to Western blotting by using anti-transcription factor EB (anti-TFEB) and anti-histone H3B (Abcam, Cambridge, United Kingdom) antibodies.