Supersignal west dura extended duration substrate chemiluminescence kit

Proteins were separated on 12% Tris-glycine gels and transferred to a Hybond-N nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% skimmed milk powder in phosphate buffered saline (PBS) for 1 hour, then incubated, first with primary antibodies in PBS with 0.05% Tween (PBST) for 1 hour and then with secondary antibodies in PBST for 45 min. Proteins were detected with a SuperSignal West Dura Extended Duration Substrate chemiluminescence kit (Thermo Scientific). FLAG-tagged proteins were detected by blotting according to the instructions for anti-FLAG M2 antibody (Sigma-Aldrich). Proteins were detected with SuperSignal West Femto chemiluminescence kit (Thermo Scientific). Cathepsin X was detected by blotting according to the instructions (R&D Systems). Information regarding antibodies is presented in S1 Table.

Tissues were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.5; 140 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; 0.1% sodium deoxycholate; 0.1% SDS) containing protease inhibitors (300 μg/ml phenylmethylsulfonyl fluoride [PMSF], 1X protease inhibitor cocktail (Sigma)). Samples (20 μg of total protein) in 1X Laemmli sample buffer were resolved on a gradient 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with PBST (PBS; 0.02% Tween 20, pH 7.5) containing 5% non-fat dry milk and incubated with primary antibodies overnight. Primary antibodies used were mouse anti-CAPN11 (1:200; MA5-21265, ThermoFisher), and rabbit anti-GAPDH (1:1000; Cell Signaling). After washing with PBST, the membranes were then incubated with a secondary anti-mouse or anti-rabbit HRP-conjugated antibodies (Cell Signaling, 1:2000), and detected with SuperSignal West Dura Extended Duration Substrate chemiluminescence kit (ThermoFisher).