Western bright quantum or sirius hrp substrates

Protein analysis of whole-cell lysates was performed using the semidry Western blot technique, according to standard protocols. For each detection, 40 µg of whole protein lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gels and blotted on nitrocellulose membranes. The detection of protein bands was performed using primary antibodies, horseradish peroxidase (HRP)-coupled secondary antibodies (Supplemental Table S2), and Western Bright Quantum or Sirius HRP substrates (Advansta, Menlo Park, CA, USA). Visualization and documentation were performed with the ChemiDoc Imaging System (BioRad, München, Germany). Densitometric analyses of Western blot protein bands were performed using the multiplex band analysis tool of the AlphaView software (Proteinsimple, San Jose, CA, USA). The protein band intensities of phospho-proteins were normalized against the intensities of total ERK1/2, total AKT, total p38 MAPK, or total JNK protein, as appropriate. All other proteins were normalized against the intensity of γ-tubulin.

Protein analyses of whole liver tissue lysates were performed using the semidry Western blot technique according to standard protocols. Antibodies against α‐SMA (M0851; Dako/Agilent), desmin (ab8592; Abcam), GFAP (MAB360; Merck/Millipore), fibronectin (610078; Becton Dickinson), ITGA5 (ab150361; Abcam), ITGB1 (ab52971; Abcam), HGF (LS‐C486534; Lifespan Biosciences), and γ‐tubulin (T5326; Sigma‐Aldrich) were used. The detection was carried out using horseradish peroxidase (HRP)‐coupled goat‐anti‐mouse or goat‐anti‐rabbit antibodies (Merck/Millipore) and Western Bright Quantum or Sirius HRP substrates (Advansta). Visualization and documentation was performed with ChemiDoc Imaging System (BioRad). The intensities of Western blot protein bands were analyzed using the multiplex band analysis tool of the AlphaView software (Proteinsimple) and normalized to the intensity of γ‐tubulin bands.