Phospho y607

Western blotting was performed as previously described. For the detection of Nrf2 and HO-1 protein levels, total proteins were extracted from the SKPs and 3D skin models using the lysis and protein loading buffers. The protein concentrations were determined using Pierce BCA Protein Assay. The Bis-Tris Gel system was established, and the primary antibodies against protein nuclear Nrf2 (anti-Nrf2 polyclonal antibody, 1 : 1000, Abcam, UK), HO-1 (anti-HO-1 monoclonal antibody, 1 : 2000, Abcam, UK), phosphorylated PI3K (p-PI3K, anti-PI3K polyclonal antibody, and phospho Y607, 1 : 1000, Abcam, UK), and phosphorylated Akt (p-Akt, anti-Akt polyclonal antibody, and phospho T308, 1 : 500, Abcam, UK) were introduced. After incubation with the secondary antibody (goat anti-rabbit IgG, 1 : 5000; Abcam, UK), signals were detected and analyzed using enhanced chemiluminescence detection system (Bio-Rad Laboratories Inc., CA, USA) and Image software. Anti-β-actin polyclonal antibody (1 : 5000; Abcam, UK) or anti-GAPDH polyclonal antibody (1 : 5000; Abcam, UK) was used as internal controls.

Western blot was applied according to previous study [20 (link)]. Cells were lysed by RIPA (Thermo Fisher Scientific, USA). Total protein levels were verified by BCA kit (Thermo Fisher Scientific, USA). 30 µg of proteins was electrophoresed in 15% SDS–PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Netherlands). The membrane was blocked with 5% skimmed milk for 2 h. Subsequently, the membrane was incubated with the primary antibodies including anti-MMP2 (ab92536, 1:1000, Abcam, USA), -MMP9 (ab38898, 1:1000, Abcam, USA), AKT (ab8805, 1:10,000, Abcam, USA), -PI3K (ab32089, 1:1000, Abcam, USA), -p-AKT (phospho S474, 1:1000, Abcam, USA), and -p-PI3K (phospho Y607, 1:1000, Abcam, USA) for 1 h at indoor temperature. Furthermore, the membrane was incubated with secondary antibody (ab6721, 1:2000, Abcam, USA) conjugated with HRP for 45 min at indoor temperature. Then, ECL Western blotting kit (Santa Cruz Biotechnology, Inc.) was adopted for membrane stain and analyzed with ImageJ software. Each experiment was repeated three times.