Anti rankl

Cells were harvested, lysed with lysis buffer (ASPEN, Wuhan, China), and
centrifuged at 16,200 g for 10 min. Total proteins in the supernatant were
measured using a BCA protein assay kit (ASPEN, Wuhan, China). Total proteins
were extracted from MC3T3-E1 cells. The protein at 30 µg was resolved by 10%
SDS-PAGE gels (ASPEN, Wuhan, China). After electrophoresis, the proteins were
transferred onto nitrocellulose membrane (Millipore, USA). The membranes were
blocked with 5% nonfat milk (ASPEN, Wuhan, China) at room temperature for 1 h.
The samples were probed with anti-GAPDH (1:10,000; Abcam, UK), anti-IL-1β
(1:1000; Abcam, UK), anti-TNF-α (1:500; Abcam, UK), anti-RANK (1:1000; Abcam,
UK), anti-RANKL (1:500; Novusbio, Shanghai, China), anti-OPG (1:1000; Abcam,
UK), anti-OCN (1:500; Abcam, UK), anti-TLR2 (1:1000; Abcam, UK), and anti-TLR4
(1:500; Abcam, UK) Abs. HRP-conjugated goat anti-rabbit IgG (1:10000; ASPEN,
Wuhan, China) was used for detection.

Western blot analysis was performed as described previously ^{63 (link)}, using the following antibodies: anti-NGFR, Cell Signaling, catalog # 8238S; anti-phospho-NF-κB p65 (Ser536), Cell Signaling, catalog #3033; anti-NF-κB p65 (D14E12), Cell Signaling, catalog #8242; anti-phospho-IκBα (Ser32), Cell Signaling, catalog # 2859; anti-IκBα, Novus Biologicals, catalog # NB100–56507; anti-p-SMAD1, Cell Signaling, catalog # 9516S; anti-SMAD1, abcam, catalog # ab63356; anti- Phospho-IKKα (Ser176)/IKKβ (Ser177), Cell Signaling, catalog #2078; anti-IKKβ, Cell Signaling, catalog #2678; anti-β-actin, Sigma-Aldrich, catalog # A5441; anti-RANKL, Novus Biologicals, Clone 12A668.