Anti cathepsin b

JAWS II and GM-BM cells were seeded on coverslips placed in a 24-well plate at a density of 1.5 × 10⁵ cells per well. Cells were left uninfected or were treated with ECTV for 60 min. at 37 °C. After 4, 12 and 24 h, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St Louis, MO, USA) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS. Next, JAWS II and GM-BM cells were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in 0.1% Triton X-100 and incubated for 45 min. with anti-cathepsin B, anti-cathepsin L (both from Abcam, Cambridge, MA, USA) and anti-cystatin B (Thermo Fisher Scientific) primary antibodies. After washing with 0.1% Triton X-100 in PBS, cells were incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with rhodamine Red-X (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) diluted in blocking solution for 1 h. ECTV antigens were stained with FITC-conjugated polyclonal antibodies for 1 h. Viral and nuclear DNA was stained with Hoechst 33342 (Sigma-Aldrich) solution for 10 min. in the dark. Slides were mounted in ProLong Gold Antifade Reagent (Life Technologies). Images were captured using Olympus BX60 fluorescence microscope and analyzed with Cell^F software (Soft Imaging System, Olympus, Tokyo, Japan) and ImageJ (NIH, Bathesda, MD, USA).

MG132 and lactacystin were obtained from Calbiochem (Darmstadt, Germany; 474790 and 426100). Chloroquine, 3MA, 3NP, doxorubicin, cycloheximide and etoposide were obtained from Sigma-Aldrich China (Shanghai, China; C6628, M9281, N5636, D1515, N11534 and E1383). The antibodies used were as follows: anti-DRAM1 (Abcam, Cambridge, MA, USA; ab58807), anti-BAX (Abcam; ab5714), anti-Bcl-2 (Santa Cruz, Dallas, TX, USA; sc-7382), anti-Atg5 (Cell Signaling, Beverly, MA, USA; 2630), anti-caspase-3 (Santa Cruz; sc-271028), anti-cathepsin B (Abcam; ab49231), anti-BID (Cell Signaling; 2002), anti-Lamp2 (Santa Cruz; sc-20004), anti-cytochrome c (Santa Cruz; sc-7159), anti-HSP60 (Santa Cruz; sc-65568), anti-p62 (Abcam; ab96134), anti-LC3 (MBL, Nagoya, Japan; PD015 ) and anti-β-actin (Santa Cruz; sc-47778).