Runx2 8486

hPDLCs (hPDLCs1 and hPDLCs2) were cultured in 60-mm dishes for 1 week, starved for 12 h after reaching 90% confluence, and then treated with APN three times (once every 2 days) during the culture period. Total protein was extracted using RIPA buffer (CW2333S; Applygen, Beijing, China) and the concentration was measured with bicinchoninic acid reagent (Thermo Fisher Scientific, MA, U.S.A.). Protein samples (40 mg/lane) in loading buffer were separated by SDS/PAGE and transferred on to a polyvinylidene difluoride membrane (Millipore, MA, U.S.A.). The primary antibodies used were as follows: anti-GAPDH (g0314; Santa Cruz Biotechnology, California, U.S.A.), anti-runt-related transcription factor 2 (RUNX2) (8486S; Cell Signaling Technology (CST), MA, U.S.A.), anti-P-p38 (4511; CST), anti-p38 (8690; CST), anti-P-p65 (3033; CST), anti-p65 (3034; CST), anti-APPL1 (3858S; CST) at 1:1000. Anti-rabbit secondary antibody (7074S; CST) at 1:5000 was used as described previously [16 (link)]. The Western blots were scanned using an Odyssey® CLx Infrared Imaging System to visualize the protein bands and were analyzed using ImageJ software (National Institutes of Health).

Protein lysates were collected, and the total protein concentration was determined using a BCA protein assay, as previously reported [6 (link)]. Protein samples were prepared by adding 1^x Laemmli’s sample buffer with 2-mercaptoethanol, heated for 5 min at 99 °C and separated by SDS-PAGE electrophoresis, followed by transfer onto a PVDF membrane. Precision Plus Protein Standards (161-0376, Bio-Rad Laboratories, Hercules, CA, USA) or PageRuler™ Plus Prestained Protein (PI26620, Thermo-Fisher Scientific) were used. Then, the primary (RUNX2 (8486) Cell Signaling Technology, Danvers, MA, USA; beta-Catenin (PA5-19469), Thermo Fisher Scientific; Aggrecan (MA3-16888), Invitrogen by Thermo Fisher Scientific; Thermo Fisher Scientific; β-actin (BA3R), Thermo Scientific; ERK (137F5), Cell Signaling; p-ERK (D13.14.4E), Cells Signaling) and secondary (Anti-mouse (7076) Cell Signaling; Anti-rabbit (7074) Cell Signaling) antibodies were incubated with PVDF membranes, and chemiluminescent signal acquisitions were obtained with the Uvitec Imager. ImageQuant software (GE Healthcare, Little Chalfont, UK) was used to perform densitometric analyses, as previously reported [22 (link)].