M per buffer

EPT3M1-STAT3 cells were lysed with cold M-PER buffer (Thermo Fisher Scientific, MA, United States, Pierce cat. no. 78501) containing protease (Roche, Basel, Switzerland, cat. no. 11836153001) and phosphatase inhibitors (Thermo Fisher Scientific, MA, United States, Pierce cat. no. 78420) and centrifuged (18,000 × g for 10 min at 4°C). Lysates were diluted to the same final volume and proteolyzed in TNC buffer [50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 10 mM CaCl2]. Then, 200 µM 323–1 or 200 µM 323–2 or the same volume of DMSO was added and incubated for 1 h at room temperature (RT). A measure of 1.25 mg/ml of pronase solution was diluted serially using 1x TNC buffer to generate 1:300, 1:1,000, and 1:3,000 pronase stock aliquots. Pronase was added into both the DMSO and drug groups and incubated for 30 min at RT. Digestion was stopped by adding 4X loading buffer and heating to 90°C for 10 min immediately prior to the Western blot assay, according to publications (Lomenick et al., 2009 (link); Lomenick et al., 2011 (link)).

At 24 h p.i., virus-infected cells were washed with PBS and lysed using M-PER buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 0.5% protease inhibitor cocktail (Pierce, Rockford, IL, USA). The cell lysates were clarified by centrifugation, and the total protein content was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a PVDF membrane. Viral proteins were detected using primary antibodies specific for DENV NS3, DENV E, DENV prM, ZIKV E, and ZIKV prM followed by a horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit secondary antibody. As a loading control, cellular β-actin was detected with an anti-β-actin-specific primary antibody and HRP-conjugated goat anti-mouse secondary antibody. After the addition of a chemiluminescent HRP substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce), images were obtained using a LAS-4000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).

Protein extracts from bone were prepared in M-PER buffer (cat#78501, Thermo Fisher Scientific, USA) containing phosphatase and proteinase inhibitors (cat#78442, Halt^™ Protease and Phosphatase Inhibitor Single-Use Cocktail, Thermo Fisher Scientific, USA). After surgical removal of the epiphysis and flushing of the marrow, the bone shafts of femurs and tibias were snap frozen in liquid nitrogen and then homogenized with a Precellys homogenizer (Bertin Instrument, France). Proteins were separated by electrophoresis in NuPAGE^™ 4 to 12% Bis-Tris Mini Protein Gels (Thermo Fisher Scientific, USA), and then transferred to Immobilon^®-FL PVDF Membrane (Millipore, USA). The membranes were blocked with Odyssey Blocking Buffer (PBS) (LI-COR Biosciences, USA), and then incubated with primary antibodies overnight. The primary antibodies included Anti-Glucose Transporter Glut1 antibody [EPR3915] (ab115730, 1:2000), Anti-Hexokinase II antibody [EPR20839] (ab209847, 1:1000), Anti-Lactate Dehydrogenase antibody [EP1566Y] (ab52488, 1:1000) and Anti-beta Actin antibody [AC-15] (ab6276, 1:2000). The secondary antibodies IRDye^® 800CW Donkey anti-Rabbit IgG (H + L) (LI-COR Biosciences, 1: 20,000) and IRDye^® 680RD Goat anti-Mouse IgG (H + L) (LI-COR Biosciences, 1:20,000) were used. The protein bands were imaged with Odyssey^® DLx Imaging System and quantified with Image J.