Pro preptm protein extraction kit

HCT116 cells were harvested by scraping from the wells and washed twice with cold phosphate buffered saline (PBS). Total cell extracts were prepared using a PRO-PREP^TM protein extraction kit (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do, Korea). Lysates were centrifuged at 16,000 rpm at 4 °C for 30 min to obtain the supernatants. Protein concentration was determined by a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA), loaded on 10% SDS-PAGE, and electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies overnight at 4 °C, washed three times, and reincubated with peroxidase-labeled secondary antibody for 2 h. A chemiluminescence (ECL) western blot kit (Amersham, Arlington Heights, IL, USA) was used to detect specific proteins.

To detect intracellular signaling in DCs, recombinant Enpp2-treated DCs, Enpp2 siRNA-transfected DCs (siEnppP2 DCs), Enpp2 inhibitor (PF8380)-treated DCs, imDCs, and mDCs were lysed using the Proprep^TM protein extraction kit (Intron Biotechnology, Gyeonggi, Korea) in the presence of a serine/threonine phosphatase inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein (10 μg) were separated on SDS-PAGE gels, and transferred to PVDF membranes. The membranes were blocked with 5% (w/w) skim milk in TBST, and then incubated with primary antibodies specific for pErk1/2, Erk1/2, p-JNK, JNK2, pp38, p38, NF-κBp65, or GAPDH (all diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with an HRP-conjugated secondary antibody (diluted 1:2000; Cell Signaling Technology). To detect Enpp2, a culture supernatant was concentrated by ultracentrifugation. Proteins in samples were separated and transferred to PVDF membranes prior to detection with an anti-ENPP2 antibody (Cayman Chemical, Ann Arbor, MI, USA) and an HRP-conjugated anti-rabbit antibody (Abcam, Cambridge, MA, UK). The membranes were exposed to ECL reagents (Thermo Scientific), and signals were detected using a Luminescent image analyzer (LAS-4000; Fujifilm, Tokyo, Japan).