Hrp conjugated horse anti mouse

Cells were seeded onto 6-well plates (Corning) and allowed to adhere overnight. After treatment, cells were lysed with ice-cold lysis buffer (50 mM Tris-base, 40 mM sodium pyrophosphate,100 μM sodium fluoride, 150 mM sodium chloride, 1% Triton X-100, 10 mM EGTA and EDTA sodium salt, 1x complete EDTA-free protease inhibitor, 1× phostop (Roche, Hertfordshire, UK). Lysates were subjected to sonication at an amplitude of 15 μm using a soniprep-150 sonicator (Sanyo, Watford, UK). Protein was quantified using a BCA assay kit and Nanodrop 2000 (both Thermo Fisher, Waltham, Massachusetts, USA).
Anti-PFKFB3, GAPDH and anti-PARP monoclonal rabbit antibodies were used at a concentration of 1:1000 for probing (#13123, #5174 and #9542S respectively Cell Signaling Technology). Mouse anti-PMCA4 (JA9, # MA1–914, Thermofisher) was also used 1:1000. HRP conjugated goat anti-rabbit antibody (#7074 Cell Signalling Technology) or HRP conjugated horse anti-mouse (#7076 Cell Signalling Technology) were used as a secondary antibody. Clarity ECL, a Chemidock and ImageLab acquisition software were used to produce digital images (BioRad). ImageJ software was used to quantify an PARP:cleaved-PARP band volumes in MIA PaCa-2, BxPC-3 and HPSCs after 6 h of treatment with 10 μM PFK15.

Protein concentrations from the cell lysates were measured as described above by BCA assay and 20μg of protein were resolved in SDS-PAGE gels and transferred to 0.2μm nitrocellulose membranes. 12% and 6% gels were used for separation of lower and higher molecular weight proteins, respectively. Rabbit anti-PSMA2 (Cell Signaling, Cat. 2455), anti-THBS2 (Abcam, Cat. ab84469), and anti–CST3 (Abcam, Cat. Ab109508), and mouse anti-ZIKV NS1 (BioFront Technologies Cat. BF-1225-06), anti-caspase 3 (Cell Signaling Cat. 3G2), anti-STAT1 (Cell Signaling, Cat. 9176S) and anti-Beta-Actin (Cell Signaling, Cat. 3700S) primary antibodies were used to detect specific proteins. (HRP)-conjugated horse anti-mouse (Cell Signaling, Cat. #7076) or anti-rabbit (Cell Signaling, Cat. #7074) secondary antibodies were used for detection of the immune complexes. Protein bands were imaged with an Alpha Innotech FluorChemQ MultiImage III after developed with ECL reagents. Band intensities were quantified with Image J 1.50i (USA) and graphically presented by Graphpad Prism software (La Jolla, California, USA).

Cell lysate was generated from tumor samples as described in the Registered Report (Li et al., 2015 (link)). Membranes were loaded with 40 µg of total protein and probed with: rabbit anti-CD44 (AbCam, cat# ab51037, clone EPR1013Y, RRID:AB_868936), 1:1000 dilution; mouse anti-ß-actin (Cell Signaling Technology, cat# 3700, clone 8H10D10, RRID:AB_2242334), 1:1000 dilution and the appropriate secondary antibody: HRP-conjugated goat anti-rabbit (Cell Signaling Technology, cat# 7074, RRID:AB_2099233), 1:2000 dilution; HRP-conjugated horse anti-mouse (Cell Signaling Technology, cat# 7076, RRID:AB_330924), 1:5000 dilution. Membranes were washed with TBST and incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, cat# 34080) according to the manufacturer’s instructions. Scanned Western blots were quantified using ImageJ software (RRID:SCR_003070), version 1.50a.