Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g

To examine which signalling pathways were affected by LY, BMMs were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. The cells were pre-treated with or without 0.4 μM LY for 2 h. Cells were then stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, 30 or 60 min. To determine the effect of LY on NFATc1, BMMs were treated with 50 ng/mL RANKL, with or without 0.4 μM LY, for 0, 1, 3 or 5 days. Total protein was extracted from cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich, St Louis, MO, USA). Lysates were centrifuged at 12,000 × g for 15 min, and the supernatants were collected. Proteins were resolved on 10% SDS-PAGE gels and transferred by electroblotting to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% nonfat dry milk in TBST (50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. Protein bands were developed using a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Abcam, Cambridge, MA, USA), followed by detection using an electrochemical luminescence reagent (Millipore, Billerica, MA, USA). Protein bands were visualized using the LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan).

CEP cells were cocultured with P. acnes supernatant or rMIF for 48 h. CEP cells were cultured with unstimulated or 5% P. acnes supernatant alone or 5% P. acnes supernatant and 20 μM 4-IPP (MIF inhibitor) for 24 hours. They were extracted using RIPA lysis buffer (Solarbio, Beijing, China) containing a protease-inhibitor cocktail. The supernatants were collected after centrifugation at 12,000 rpm for 15 min. Proteins were separated on 10% SDS-PAGE gels and were transferred by electroblotting to PVDF membranes (Hercules, CA). The membranes were blocked (1 h) in 5% (w/v) nonfat dry milk in Tris-buffered saline containing Tween (TBST). The membranes were incubated (4 °C, overnight) with primary antibodies for MIF, MMP-13, Col II, IL-6, IL-1β, β-actin, P65, p-P65, ERΚ1/2, and p-ERΚ1/2 (1:1000 dilution, Abcam) and then were incubated with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000 dilution; Abcam). Immunoreactive bands were detected using an electrochemical luminescence reagent (Millipore, Billerica, MA, USA) and were visualized using Image Lab software (Bio-Red, Hercules, CA, USA).