Ab9635

TNF-α in osteoclasts after CASC11 was detected by Western blot. Briefly, Monarch^® Total RNA Miniprep Kit (NEB) was used to extract total RNAs from osteoclasts. Following denaturing, gel electrophoresis was performed using 10% SDS-PAGE. After gel transfer to PVDF membranes, membranes were blocked in 5% non-fat milk for 2 hrs at denaturing. Membranes were then incubated with rabbit anti-human TNF-α (1:1200, ab9635, Abcam) or GAPDH (1:1800, ab8245, Abcam) overnight at 4°C. After that, incubation with goat anti-rabbit IgG-HRP secondary antibody (1:1500, MBS435036, MyBioSource) was performed for 2 hrs at room temperature. After ECL (Sigma-Aldrich, USA) method was used for signal development and signals were normalized using Image J software.

Phosphate-buffered saline was used to wash AC16 cells twice. Then, 10⁵ AC16 cells were mixed with 1 mL of radioimmunoprecipitation assay (RIPA) solution (Sigma-Aldrich) to prepare cell lysates. Protein concentrations were measured using the bicinchoninic acid (BCA) assay (Sigma-Aldrich), followed by protein denaturation in boiling water for 10 minutes. Protein molecules were separated using a 10% sodium dodecyl sulfate-PAGE gel, followed by gel transfer to polyvinylidene difluoride membranes. Blocking was performed using PBS containing 5% nonfat milk for 2 hours at room temperature. After that, membranes were first incubated with rabbit primary antibodies to GAPDH (ab9845, Abcam, Cambridge, UK) and TNF-α (ab9635, Abcam) for 12 hours at 4°C. Then, membranes were further incubated with secondary antibodies of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (IgG; ab6721, Abcam) for 2 hours at room temperature. Signals were then produced using ECL chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA), and ImageJ v1.48 software (National Institutes of Health, Bethesda, MD, USA) was used to normalize signals.

Immunohistochemistry was performed as described previously [25] (link), [26] (link), [27] (link). For immunohistochemistry experiments, frozen 8-μm-thick sections were fixed in 4% paraformaldehyde. The sections were blocked by 8% normal goat serum for 4 h and then incubated in specific primary antibodies. After being washed three times by PBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies. Staining is visualized using substrate diaminobenzidine. The following antibodies were used: α-smooth muscle actin (α−SMA, #ab7817, Abcam, Cambridge, UK, 1: 600 dilution), PCNA (clone PC10, Millipore, Milford, MA, USA, 1;1000 dilution), tumor necrosis factor-α (TNF-α, #ab9635, Abcam, Cambridge, UK, 1: 1000 dilution), interleukin-1β (IL-1β, #MAB4012, R&D Systems, Minneapolis, MN, USA, 1: 1000 dilution), CD68 (#MA5-13324, Invitrogen, Carlsbad, CA, USA, 1: 2000 dilution), 3-nitrotyrosine (3-NT, #sc-32757, 1: 4000 dilution) malondialdehyde (MDA, #ab6463, 1: 2000 dilution), myeloperoxidase (MPO, #PA5-16672, Invitrogen, 1: 2500 dilution), chemokine (C-C motif) ligand 2 (CCL2, #MA5-17040, Invitrogen, 1: 1500 dilution) and chemokine (CXC motif) ligand 2 (CXCL2, #PA5-28820, Invitrogen, 1: 2000 dilution).