Nih image j

Confluent cells were washed once with ice-cold PBS and lysed with 1x PhosphoSafe lysis buffer (Merck Millipore). Cell lysates were sonicated and centrifuged for 10 min at 14.000 × g and 4 °C following determination of total protein concentration in supernatants using BioRad DC Protein Assay. Samples containing equal amounts of protein were prepared using 3x SDS Sample Buffer and 125 mM Dithiothreitol (both from New England Biolabs), subjected to gel electrophoresis using BioRad mini-PROTEAN TGX any-kD precast gels and blotted to 0.45 μm PVDF membranes. Membranes were blocked with nonfat dry milk (Carl Roth GmbH, Karlsruhe, Germany) or BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare) were used for detection of proteins on a BioRad ChemiDoc XRS imaging system. RotiFree stripping buffer (Carl Roth GmbH) was used for membrane stripping. Signal intensities were quantified by densitometry and computed with either NIH image J or Image Lab (version 5.2.1, BioRad).

Cells were treated as indicated in text and figure legends and then lysed for 1 h using triton lysis buffer (PBS with 1% Triton X-100; 25 mM Tris, pH 7.5; and 150 mM NaCl) with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) at 4 °C. Protein abundance was measured by Bradford assay (Bio-Rad, Hercules, CA, USA). Laemelli buffer was added to samples and the tube was boiled for 5 min, and then was subjected to SDS-PAGE and transferred to PVDF membrane (Bio-Rad) [15 (link)]. Membranes were blocked as per the manufacturer’s instructions then exposed to HO-1 (Cat No. ADI-SPA-895, Enzo, Farminglade, NY, USA), NRF2 (SC-722, Santa Cruz Bio, Dallas, TX, USA), or Actin (Cat. No. A2228, Millipore Sigma, Burlington, MA, USA) antibodies overnight. Membranes were washed with tris buffered saline containing 1% Tween-20 (TBST) for 30 min followed by incubation with secondary antibodies (GE Healthcare, Pittsburgh, PA) [15 (link)]. Visualization was performed using ECL (Bio-Rad) and densitometry measured using NIH ImageJ (Bethesda, MA, USA).