Qpro bca kit standard

Proteins from the parenchymal region of each mammary gland were extracted with RIPA buffer containing 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 5 mM EDTA (Sigma), protease inhibitor cocktail and phosphatase inhibitor cocktail II (both from Sigma). Equal amounts of protein, determined by QPRO-BCA Kit Standard (Cyanagen, Bologna, Italy) according to the manufacturer’s protocol, were subjected to SDS–PAGE. Fractionated proteins were transferred to nitrocellulose filters and the uploading of equal amounts of protein per sample was confirmed by staining the blot with Ponceau S (Sigma). The expression levels of phosphorylated S6 (pS6) and β-actin were determined according to the signals generated with their respective antibodies. Signals were generated with WESTAR Supernova (Cyanagen, Bologna, Italy) combined with Super Signal chemiluminescent substrate (Thermo Fisher Scientific), and detected in a Syngene analyzer (Cambridge, UK).

Cellular pellets were vigorously resuspended in 50 μl of ice-cold RIPA lysis buffer (Merck, 20–188) supplemented with a protease inhibitor cocktail (cOmplete, Roche diagnostics GmbH, 11697498001). After 30 min incubation on ice, the lysates were centrifuged (16,000 × g; 20 min; 4°C), the cleared supernatants were transferred to fresh tubes, and protein concentrations were determined by bicinchoninic acid (BCA) method (absorbance at 570 nm), and by using the QPro-BCA Kit standard (Cyanagen, 32 PRTD1). Samples were stored at −20°C until usage. Immunoblotting and antibodies were previously described (Abu Rass et al., 2022 (link)). Densitometry of the immunoblots was quantified using the ImageJ program.

Cells or liver specimens were lysed with lysis buffer (RIPA—Sigma-Aldrich, Rehovot, Israel) containing a protease inhibitor cocktail and phosphatase inhibitors (Bimake, Houston, TX, USA). Protein levels were quantified using the QPRO-BCA kit standard (Cyanagen, Bologna, Italy). Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes (Tamar, Mevaseret Zion, Israel), and then probed with monoclonal antibodies specific for αSMA (Sigma-Aldrich, Rehovot, Israel), HSC70 (Santa Cruz Biotechnology, INC., Santa Cruz, CA USA), or CD63 (BioVision, Inc., Milpitas, CA, USA), or a polyclonal anti-actin 1–19 antibody (Santa Cruz Biotechnology, INC., Santa Cruz, CA, USA) diluted 1:1000. Peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (1:10,000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) were used as secondary antibodies. Immunoreactive bands were visualized using the Western blot chemiluminescence reagent (Cyanagen, Bologna, Italy). Relative quantification of the proteins was performed using the ImageJ software.