Horseradish peroxidase conjugated secondary antibody

Phycoerythrin-conjugated rat monoclonal anti-mouse RANK antibody (12-6612) and the isotypic antibody, phycoerythrin-conjugated rat IgG2b (15-4815), were purchased from eBioscience, Inc. (San Diego, CA, USA). Anti-phospho-AKT (Ser473) (9271), anti-AKT (9272), anti-phospho-p44/42 ERK (Thr202/Tyr204) (4370), anti-ERK (4695), and anti-signal transducer and activator of transcription 3 (STAT3) (4904) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). The antibody against mouse phospho-STAT3 (Ser727) (sc-135649) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-RANK (ab200369), anti-calcitonin receptor (CTR) (ab11042) and anti-IL-21R (ab5980) and anti-β-actin (ab8229) antibodies were obtained from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated secondary antibodies were purchased from LI-COR Biosciences (Lincoln, Nebraska, USA). Recombinant IL-21, RANKL and M-CSF were obtained from Peprotech, Inc. (Rocky Hill, NJ, USA). AG490 [Janus kinase 2 (JAK2)/STAT3 inhibitor], LY294002 (PI3K/AKT inhibitor), and PD98059 (ERK inhibitor) were supplied by Selleck Chemicals (Houston, TX, USA).

The protein expressions levels of ephrin-b2, NFATc1, TRAP, CK, MPP9 and GAPDH were measured with and without ephB4-Fc treatment. After 5 days, radioimmunoprecipitation assay (RIPA) lysis buffer (cat. no. C500005; Sangon Biotech Co., Ltd.) containing 1 µM protease inhibitor was added to 2×10⁷ BMMs in 6-well plates for 15 min. Samples were centrifuged (4°C) at 12,000 × g for 10 min and the supernatant was collected. Total protein concentration was measured by the bicinchoninic acid assay (BCA). Equal amounts of the protein lysates were separated via SDS-PAGE (10% gel) and gels were transferred to polyvinylidene difluoride membranes, blocked for 1 h with 5% (w/v) milk, and incubated at 37°C with primary antibodies against GAPDH (cat. no. #8884; 1:1,000; Cell Signaling Technology, Inc.), C-FOS (cat. no. #2250; 1:1,000; Cell Signaling Technology, Inc.), NFATc1 (cat. no. #8032; 1:1,000; Cell Signaling Technology, Inc.) and TRAP (cat. no. ab133238; 1:1,000; Abcam) overnight. The Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) was used to detect horseradish peroxidase-conjugated secondary antibodies (cat. no. #7074; 1:5,000; Cell Signaling Technology, Inc.) reactivity.

Western blotting was applied to quantify protein expression. Briefly, cells were rinsed with PBS and then lysed in extraction buffer and phenylmethylsulfonyl fluoride. Cell extracts were centrifuged at 4°C for 10 minutes, and the supernatants were then collected. The protein concentration of cells was measured using bicinchoninic acid protein quantification kit (Beyotime). The same amounts of the samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime), and then transferred to nitrocellulose membranes (Beyotime). The membranes were blocked with 5% skim milk, using PBS with 0.05% Tween-20 for 2 hours. The membranes were then probed successively with Col I, Runx2, OPN and β-actin at 4°C overnight. The membranes were washed and then incubated with the respective horseradish peroxidase-conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 hour at room temperature. Proteins were detected by autoradiography (Bio-Rad). The primary antibodies used in this study are listed in Table 2.

After the indicated treatments, Leydig cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethane sulfonyl fluoride and 1% Triton X-100). Samples were fractionated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 10% nonfat milk and incubated at room temperature with primary antibodies for 2 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Li-cor Biosciences, Lincoln).
NE, USA; catalogue no. 926-32211, 926-68021) at a dilution of 1:10,000 at room temperature for 1 h. Images were obtained using Odyssey (LI-COR Biosciences, Lincoln, NE, USA). Membranes were then stripped and re-probed with GAPDH antibody to ensure equal protein loading. TNF-α (D2D4, 1:1,000) and AMPK (catalogue no. 23A3, 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Interleukin (IL)-1β antibody (catalogue no. CC36131) at a dilution of 1:1,000 was purchased from Bioworld Technology (Nanjing, China). Odyssey infrared imaging system (version 3.0.21; LI-COR Biosciences, Lincoln, NE, USA) was used for densitometry.