β actin sc 130656

Protein A/G beads, anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, mouse monoclonal or rabbit polyclonal antibodies specific for CCN3 (SC-136967), Twist (SC-81417), N-cadherin (ab76057), E-cadherin (ab40772), p-FAK (SC-11765-R), FAK (SC-932), p-Akt (SC-16646-R), Akt (SC-5298), HIF-1α (ab1), and β-actin (SC-130656) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Abcam (Cambridge, UK). Recombinant human CCN3 was purchased from PeproTech (Rocky Hill, NJ, USA). All other chemicals were obtained from Sigma–Aldrich (St Louis, MO).

Western blot was performed according to the method described previously [29 (link)] to assess Keap-1/Nrf-2/HO-1 and p38 MAPK/NF-κB pathways’ protein expression. Briefly, renal tissue samples were mixed with RIPA buffer containing protease inhibitor; the extracted protein was measured using a Nano Drop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, 50 μg of the total extracted protein was separated via SDS-PAGE and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in TBS enclosing 3% bovine serum albumin and 0.1% Tween 20 for one hour at room temperature. PVDF membranes were washed (TBS containing 0.1% Tween 20) and incubated first with a 1:1000 dilution of the primary antibodies (Keap-1, Nrf2, HO-1, P38 MAPK and NF-κB) for two hours and, then, with a 1:5000 dilution of the secondary antibody at room temperature. Keap-1 (sc-514914), Nrf2 (sc-722), HO-1 (sc-136960), P38 MAPK (sc-7973), NF-κB p65 (sc-8008), reference gene (β-actin; SC-130656) and goat anti-rabbit immunoglobulin (Ig) G-horseradish peroxidase (HRP) (sc-2030) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The chemiluminescence produced was detected with the C-DiGit chemiluminescence scanner (LI-COR, Lincoln, NE, USA), and the band intensity was analyzed using the scanner software.

Neocuproine, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha medium (α-MEM), fetal bovine serum (FBS), 2,5-diphenyltetrazolium (MTT), Triton X-100, RANKL, sodium tartrate, p-nitro-phenylphosphate (PNPP), fluoresceinamine-labeled chondroitin sulfate (FACS), phosphate-buffered saline (PBS, pH 7.4), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), dimethylsulfoxide (DMSO), chlorogenic acid, caffeic acid, isoquercitrin, and isochlorogenic acid A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyeroside was purchased from Hwz Analytic GmbH (Ruelzheim, Germany). Scoparone was obtained from Phytolab GmbH&Co (Vestenbergreuth, Germany). Antibodies and protein A-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), including anti-TRAF6 (sc-7221), V-ATPase (sc-20946), and β-actin (sc-130656). A bone resorption assay kit was purchased from CosMo Bio Co., Ltd. (Tokyo, Japan). MC3T3-E1 subclone 4 and RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).