Tfeb antibody

Mice testes or cultured cells were homogenized in RIPA lysis buffer (Thermo Fisher Scientific, USA) containing protease inhibitor cocktail (Roche, USA) on ice for 30 min. Then centrifuged at 12000 g, 10 min, 4 °C. The proteins in the supernatant were collected and the protein concentrations were determined by the BCA Protein Assay Kit (Thermo Fisher Scientific).
Protein samples (20 μg) were separated by using 8–16% denaturing polyacrylamide gels, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) by using a semi-dry transfer apparatus (Bio-Rad, USA). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and immunoblotting was performed overnight at 4 °C with the TFEB antibodies (1:5000 dilution; Santa cruz) or β-actin antibodies (1:4000 dilution; Cell Signaling Technology, USA), followed by incubation with secondary antibody conjugated to HRP (Jackson ImmunoResearch, USA). Signals were generated by enhanced chemiluminescence (Millipore) and detected by luminescent image analyzer (GE imagination LAS 4000, USA).

EndoC‐βH1 cells were seeded in 96‐well plates and incubated for 24 h. Cells were then treated as indicated, rinsed with PBS once, fixed for 10 min with 4% paraformaldehyde, and stained with TFEB antibodies (Cat# 4240, Cell signaling) and DAPI. For the acquisition of the images, at least 10 fields were acquired per well of the 96‐well plate by using confocal automated microscopy (Opera High Content System; Perkin‐Elmer). A dedicated script (Medina et al, 2015 (link)) was used for analysis of TFEB localization on the different images (Harmony and Acapella software; Perkin‐Elmer). The script calculates the ratio value resulting from the average intensity of nuclear TFEB fluorescence divided by the average of the cytosolic intensity of TFEB fluorescence. P‐values were calculated on the basis of mean values from independent wells.

3.5 × 104 HeLa cells were seeded in a 24-well plate and treated overnight with DMSO or EDME 1 µM. The day after, cells were treated for 3 h in full media (Fed) or HBSS supplemented with 10 mM HEPES (nutrient starved) in presence of DMSO or EDME 1 µM. Cells were fixed in PFA 4% 10′ and permeabilized 7´ with PBS 1X and 0.02% Triton-X. TFEB antibody (Cell Signaling Cat. No. 4240, 1:100 overnight) and Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor 488 (ThermoFisher, 1:400 45´) were applied in blocking buffer saponin (1% BSA, 0.05% saponin and 50 mM NH4Cl in PBS1X). Samples were examined under a Zeiss LSM 880 confocal microscope. Optical sections were obtained under a 40 × immersion objective at a definition of 1024 × 1024 pixels (average of 8 scans), adjusting the pinhole diameter to 1 Airy unit for each emission channel to have all the intensity values between 1 and 254 (linear range). TFEB nuclear and cytoplasmic intensity was measured on unsaturated images using ImageJ software (NIH). The value reported is a ratio value resulting from the average intensity of nuclear TFEB fluorescence divided by the cytosolic intensity of TFEB fluorescence.