Mouse anti cathepsin c

Cells were washed twice with an ice-cold PBS and lysed in NP-40 lysis buffer supplemented with protease inhibitor cocktail (APExBIO). Lysates were centrifuged at 16000g at 4°C for 20 minutes to obtain post-nuclear cell fraction. Protein concentration was determined with Pierce BCA protein assay kit (ThermoFisher Scientific). Non-reducing SDS-PAGE was performed, and separated proteins were transferred to nitrocellulose membrane. Membranes were blocked for 1 hour in 5% non-fat dry milk in PBS. Membranes were incubated in primary antibodies overnight at 4°C and HRP conjugated secondary antibodies for 1h at room temperature. Bands were visualized with Clarity Max Western ECL substrate (BioRad). Images were acquired with ChemiDoc ML imaging System (Biorad). The following primary antibodies were used: mouse anti-granzyme B (sc-8022, Santa Cruz Biotechnology), mouse anti-cathepsin C (sc-74590, Santa Cruz Biotechnology), mouse anti-perforin-1 (sc-136994, Santa Cruz Biotechnology). We used anti-mouse HRP conjugated secondary antibodies (405306 BioLegend). Stain free technology (BioRad) was used for loading control.

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).

For Western blotting, the following antibodies were used: rabbit anti-cystatin F (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-N-terminal part of cystatin F (Amsbio, Abingdon, UK), rabbit anti-β-actin (Sigma-Aldrich), rabbit and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, Rosemont, IL, USA), rabbit anti-C/EBPα, mouse anti-cathepsin C, mouse anti-granzyme A, mouse anti-granzyme B, mouse anti-perforin, and mouse anti-legumain (B-8) (all from Santa Cruz Biotechnology, Dallas, TX, USA). The mouse anti-cathepsin H antibody 1D10 [87 (link)] and sheep anti-cathepsin L antibody [88 (link)] were prepared as described previously. The following secondary antibodies were used: anti-rabbit, anti-mouse, and anti-sheep secondary antibodies conjugated with horseradish peroxidase (HRP) (Jackson Immuno Research, West Grove, PA, USA); anti-rabbit and anti-mouse secondary antibodies conjugated with the fluorescent dyes DyLight 650 and DyLight 550, respectively (Invitrogen, Carlsbad, CA, USA); and anti-mouse secondary antibody conjugated with fluorescent dye StarBright 700 (Bio-Rad, Hercules, CA, USA).