Western Blot Analysis of Protein Expression

The lens was removed from enucleated eyes and the NR and RPE frozen in liquid nitrogen. Tissues were homogenised in SDS lysis buffer [100 mM Tris-HCl, pH 6.8; 2% (w/v) SDS] containing both protease and phosphatase inhibitors (Complete Mini & PhosSTOP, Roche) using a TissueLyser (Qiagen). The concentration of the soluble protein fraction was measured using the BCA Protein Assay Kit (Fisher Thermo Scientific). Immunoblotting was performed on protein extracts (6 µg) using Criterion TGX gels and nitrocellulose membranes (Bio-Rad) (Mahmood and Yang, 2012 (link)). Reactive proteins were visualised using SuperSignal West Dura Extended Duration Substrate (Fisher Thermo Scientific). Quantification was performed using a ChemiDoc MP Imaging System (Bio-Rad). The following antibodies and dilutions were used: Hamartin (1:1000, Cell Signaling Technology, 4906), S6 (1:2000, Cell Signaling Technology, 2217), pS6^S235/236 (1:1000, Cell Signaling Technology, 4857), pS6^S240/244 (1:2000, Cell Signaling Technology, 5364) and GAPDH (1:30,000, Cell Signaling Technology, 2118). All immunoblotting antibodies have been verified in a previous study (Jones et al., 2015 (link)).