Western Blot Analysis of Glycolytic and Cell Signaling Proteins

As previously reported^{6 (link)}, cells were lysed in 50 μl of PRO-PREP Protein Extraction Solution (iNtRON Biotechnology; Seongnam-si, South Korea), homogenized using a 30-gauge needle, incubated for 30 min at 4 °C, and then centrifuged at 15,000 × g in a Centrigue 5810 R (Eppendorf, Hamburg, Germany). After quantifying proteins in the extracts using the Bradford method, 20 μg protein was subjected to electrophoresis on 10% polyacrylamide gels in Tris/glycine (Invitrogen^®, Carlsbad, CA), transferred to a PVDF membrane (Millipore Corporation, Billerica, MA), and then probed with primary antibodies against ENO2, HK2, PFKFB3 (Abcam; Cambridge, UK, 1:000), JNK, p-JNK, c-Jun, p-c-Jun, and beta-Actin (Santa Cruz Biotechnology, Dallas, TX, 1:000). Primary antibodies were detected by horseradish peroxidase (HRP)-conjugated secondary antibodies (Invitrogen^®, Carlsbad, CA, 1:10000) and visualized using enhanced chemiluminescence reagents (Santa Cruz Biotechnology, Dallas, TX). The intensity ratio (IR) of western blot bands was measured using Image J 1.50i⁴⁹ .