Visualizer kit

Liver tissues were homogenized in a lysis buffer (100 mM NaCl, 50 mM Tris at pH 7.4, 1 mM EDTA, 1% Triton X-100 with protease inhibitor and phosphatase inhibitor cocktails, Roche) and denatured in a 2% SDS sample buffer (50 mM Tris at pH 6.8, 2% SDS, 100 mM dithiothreitol and 10% glycerol) for 10 min at 100 °C. The extracted proteins were separated by SDS–polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% (w/v) nonfat dried milk, probed with an antibody and detected with a Visualizer Kit (WBKLS0500, Millipore, Burlington, MA, USA). The following antibodies were used for Western blotting: S-sulfonated cysteine (ADI-OSA-820; Enzo Life Sciences, Ann Arbor, MI, USA), 3-nitrotyrosine (ab61392, Abcam, Cambridge, UK) and Gapdh (MAB374, Millipore, Burlington, MA, USA).

Tissue samples were homogenized in RIPA buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% deoxycholate) with complete protease inhibitor cocktail (04693132001, Roche) and denatured by boiling for 5 min. The extracted proteins were separated on SDS–polyacrylamide gel and electrotransferred to an Amersham™ Hybond® P Western blotting membrane, PVDF (10600023, GE Healthcare). The membranes were blocked with 5% (w/v) nonfat dry milk and 5% (w/v) bovine serum albumin (BSA, A7906, Sigma), incubated with primary antibody, washed, and then detected using a Visualizer Kit (WBKLS0500, Millipore). The antibodies used for detecting the target proteins are listed in Supplementary Table 5. Hsp70 was chosen as an internal control for each sample, and protein levels are expressed as the relative fold changes. All blots were derived from the same experiment and were processed in parallel. Un-cropped images of all blots in this study can be found in Supplementary Fig. 6.

Skin tissue and cell samples were homogenized in RIPA lysis buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 0.5% Sodium deoxycholate, 0.1% SDS and 1% Triton X-100 with complete protease inhibitor and phosphatase inhibitor cocktails [Roche]) and then denatured in SDS sample buffer (50 mM Tris at pH 6.8, 100 mM dithiothreitol, 2% SDS and 10% glycerol) for 10 min at 100 °C. The extracted proteins were separated by SDS–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), followed by electro-transferred to a polyvinylidene fluoride membrane (PerkinElmer, Waltham, MA, USA). The membranes were blocked with 5% (w/v) non-fat dried milk solution, then incubated with the required specific antibody. This was followed by detection by visualizer kit (Millipore, Burlington, MA, USA, WBKLS0500). The following antibodies were used for the Western blotting: Cisd2 [29 (link)], Gapdh (Millipore, MAB374), β-tubulin (Millipore, 05661) and MMP-1 (Proteintech, Rosemont, IL, USA, 10371-2-AP).