P jnk

Western blots were performed as described from our previous studies (Campolo et al., 2013 (link)).
Specific primary antibodies, anti-IkB-α (1:500; Santa Cruz Biotechnology), anti-NF-κB p65 (1:500; Santa Cruz Biotechnology), anti- p-JNK (1:500; Santa Cruz Biotechnology) anti-iNOS (1:500; BD Transduction), anti-p-P38 (1:1000; Cell signaling) and anti-MnSOD (1:500 Millipore) in 1x PBS, 5%(w/v) non-fat dried milk, and 0.1% Tween 20 were used at 4°C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000; Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h at room temperature. The levels of β-actin (1:2000; Santa Cruz Biotechnology) and lamin A/C (nuclear fraction 1:500 Sigma–Aldrich, Milan, Italy) served as an internal control for protein loading. The relative expression of the protein bands of IkB-α (37 kDa), NF-κB p65 (65 kDa), iNOS (130 kDa), p-JNK (46 kDa), p-P38 (38 kDa), MnSOD (24 Kda) were detected with an enhanced chemiluminescence (ECL) system (Thermo, USA) and visualized with the Chemi Doc XRS (Bio-Rad, USA) and analyzed by using Image Lab 3.0 software (Bio-Rad, USA).

Protein extraction was performed by lysing the cells in 2 × Laemmli sample buffer. Proteins were separated by SDS gel electrophoresis using 10% polyacrylamide gels and transferred onto nitrocellulose membranes ER (Bio-Rad Laboratories). Nonspecific binding was blocked by TBS-Tween with 5% nonfat dry milk. Transfer membranes were immunoblotted with the indicated antibodies: RIPK1 (BD Biosciences), RIPK3 (Cell Signaling), MLKL (Sigma-Aldrich), pMLKL (Cell Signaling), TAK1 (Cell Signaling), GSK3ß (Cell Signaling), Aurora A (Cell Signaling), p38 (Thermo Fischer Scientific), pErk (Thermo Fischer Scientific), pJNK (Thermo Fischer Scientific), pIκß (R&D Systems) and β-actin (Sigma-Aldrich) all diluted 1:1000. Anti-rabbit (GE Healthcare), anti-mouse (GE Healthcare) or anti-rat (Sigma-Aldrich) antibodies conjugated to horseradish peroxidase were used as the secondary antibodies.

Nitrogen-containing BP, alendronate sodium salt trihydrate (FOS) was purchased from Wako in Japan. SCGMTM BulletKitTM, N-acetyl-cysteine (NAC), fluorescein isothiocyanate (FITC)-conjugated annexin V, and ECL chemiluminescence were acquired from Sigma in China. PTHrP, cell-permeable 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), U0126 (inhibitor of ERK1/2), SB203580 (inhibitor of p38), sanguinarine chloride (inhibitor of MKP1), lipofectamine LTX transfection reagent, Tris HCl, NaCl, EDTA, 1% Triton X-100, 1% sodium hydroxide, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail, cocktail Set II, bovine serum albumin (BSA), Considering bicinchoninic acid (BCA), nitrocellulose membrane were obtained from ThermoFisher in China. In addition, α-Tubulin, peroxidase-conjugated goat anti-rabbit IgG and the antibodies of p-ERK1/2, ERK1/2, p-Akt, Akt, p-p38, p38, p-JNK, JNK, MKP-1, and p-p66^Shc (Ser36) were acquired from ThermoFisher in China. Propidium iodide was purchased from Solarbio in China.