Pxi chemiluminescence detection system

Cells isolated by centrifugation (600 xg, 2 min) were lysed in cold medium containing protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail) and the supernatant (900 xg, 15 min) was used for western blotting. Proteins were separated (4%–8% RunBlue SDS gel, Expedeon, San Diego, CA), transferred to a polyvinyl difluoride (PVDF) membrane using an iBLOT gel-transfer system (ThermoFisher), blocked in Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.2% Tween-20 and 5% BSA for 1 hr, washed in the same medium, and incubated with primary antibody (16 hr, 4°C) in the blocking buffer. After washing (3 × 5 min), the membrane was incubated with secondary antibody (1 hr, 20°C), washed (3 × 5 min). Bands were visualized using ECL Prime western blotting detection reagent and a Syngene Pxi chemiluminescence detection system with GeneTools software.

Cells in 6-well plates were washed, lysed in RIPA buffer (30 μl, 4°C, 1 h) by using a syringe needle and sonication, and the supernatant (14,000 g, 15 min, 4°C) was used for analysis.
RIPA buffer comprised: 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8. Protein samples were separated on 4–12% Bis-Tris PAGE gels, transferred to a polyvinyl difluoride (PVDF) membrane using an iBLOT system, and blocked by incubation in TBST with gentle shaking (1 h, 20°C). Tris-buffered saline (TBS) comprised: 150 mM NaCl, 50 mM Tris pH 7.5. For TBST, TBS was supplemented with 0.1% Tween-20 and 5% BSA. The membrane was washed with TBST, incubated with primary antibody (16 h, 40°C), washed with TBST (3×5 min, 20°C), incubated with HRP-conjugated secondary antibody in TBST with 1% BSA (1 h, 20°C), and washed with TBST (3×5 min, 20°C). Enhanced chemiluminescence (ECL) primer western blotting detection reagent (Amersham, UK) and a Syngene PXi chemiluminescence detection system were used to detect HRP. The antibodies used were mouse anti-cathepsin C (Santa Cruz Inc., Cat# s74590, 1:500), rabbit anti-TMCO1 (Sigma, Cat# AV49429, 1:1000), mouse anti β-actin (Cell Signaling, Cat# 8H10D10, 1:1000), donkey anti-mouse IgG-HRP (Santa Cruz Inc., Cat# sc-2314, 1:2000) and donkey anti-rabbit IgG-HRP (Santa Cruz Inc., Cat# sc-2313, 1:5000).