Coomassie blue reagent

The determination of β-galactosidase activity was carried out at 30 °C using 22 mM of o-nitrophenyl-β-d-galactopyranoside (oNPG) in 50 mM of sodium phosphate buffer (pH 6.5) as substrate, as previously described [46 (link)]. The reaction was initiated by adding 20 µL of enzyme solution to 480 µL of the substrate solution, and then incubated for 10 min using an Eppendorf thermomixer compact (Eppendorf, Hamburg, Germany) with an agitation of 600 rpm. The reaction was stopped by adding 750 µL of 0.4 M of Na₂CO₃. The release of o-nitrophenol (oNP) was measured by determining the absorbance at 420 nm. One unit of oNPG activity was defined as the amount of enzyme releasing one µmol of oNP per minute under the described conditions. Protein was determined by the method of Bradford [47 (link)] with the BioRad Coomassie Blue reagent (Marnes-la-Coquette, France) using bovine serum albumin as the standard.

Cells were lysed in buffer containing 50 mM Tris-HCl pH = 7.5, 120 mM NaCl, 1 mM EGTA, 6 mM EDTA, 15 mM Na₄P₂O₇, 20 mM NaF, and 1% Triton X-100 protease inhibitor cocktail. Lysates were clarified by centrifugation at 10,000× g for 15 min at 4 °C, and supernatants were quantified for protein content (Coomassie Blue reagent, Bio-Rad, Hercules, CA, USA). All passages were carried out on ice as described by Rapizzi et al. (Rapizzi et al., 2014). Proteins were separated by 8% or 12% sodium dodecyl sulfate polyacrylamide (SDS/PAGE) gels and transferred to PVDF membranes (Fisher Scientific or Immobilon, Millipore, MA, USA). Primary antibodies against alpha-tubulin (#3873), cyclin D1 (#55506), phospho-AMPK (Thr172) (#2535), phospho-Erk1/2 (#9106s), Erk1/2 (#9102s), N-Cadherin (#14215s), and E-Cadherin (#14472s), were from Cell Signaling Technology. Phospho-Akt (Ser473) (sc7985), Akt (sc8312), and all the secondary HRP conjugated antibodies (anti-mouse, sc-2005, and anti-rabbit, sc-2004) were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA).
Protein bands were detected with ECL reagents (Immobilon Crescendo, Millipore, Burlington, MA, USA). ImageJ software was used for the densitometric analysis of the bands.
The original whole blot (uncropped blots) showing all the bands are reported in the Supplementary Materials.

Protein concentration was estimated for left kidney homogenates by the Bradford (1976) (link) method, using Coomassie Blue reagent (1:5; Bio-Rad, Brazil). Absorbance (595 nm) values were read at the Infinite F-200 (Tecan; Grödig, Austria). Calculation was based on a bovine serum albumin standard curve; values were expressed in mg/ml.