Phosphor mapk family antibody sampler kit

Recombinant mouse soluble RANKL (Gene ID: 21943) was obtained from R&D Systems (Catalog number: 462-TEC-010, Oakville, ON, Canada). Dulbecco modified Eagles medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/mL), and TRIzol Reagent were purchased from Thermo Fisher Scientific (Burlington, ON, Canada). Ovotransferrin (conalbumin from chicken egg white) with purity ≥98% was purchased from Sigma-Aldrich (Catalog number: C0755, St. Louis, MO, USA). The tartrate-resistant acid phosphatase (TRAP)-staining kit was obtained from Sigma-Aldrich (Catalog number: 387A-1KT, St. Louis, MO, USA). The annexin V-FITC Apoptosis Staining/Detection Kit was purchased from Abcam (Catalog number: ab14085, Toronto, ON, Canada). The NF-κB pathway sampler kit (Catalog number: 9936T), MAPK family antibody sampler kit (Catalog number: 9926T), and phosphor-MAPK family antibody sampler kit (Catalog number: 9910T) were purchased from Cell Signaling Technology (Whitby, ON, Canada). Recombinant anti-TRAF6 antibody (Catalog number: ab33915), anti-c-Fos antibody (Catalog number: ab190289), and anti-α tubulin antibody (Catalog number: ab7291) were purchased from Abcam (Cambridge, MA, USA). NFATc1 antibody (Catalog number: 7A6) and cathepsin K antibody (Catalog number: E-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

The lower right lung lobes were removed and stored in liquid nitrogen for protein analysis. Briefly, the lung tissues were lysed on ice in a RIPA (P0013B, Beyotime) buffer containing 1 mM PMSF (Beyotime). After sonication, the protein concentration in the supernatants was measured using a bicinchoninic acid protein assay kit (Beyotime). Equal amounts of protein were loaded in 10% SDS–PAGE, and then the proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% bovine serum albumin for 1 h at room temperature. After rinsing in PBST 5 times, the membranes were incubated with each primary antibody overnight at 4°C: the anti-claudin 5 (Abcam, ab15106), the phosphor-MAPK family Antibody sampler kit (cell-signaling technology, #9910) the MAPK family Antibody sampler kit (cell-signaling technology, #9926), and β-actin (cell-signaling technology, #4970). After washing with PBST, the membranes were finally incubated with the appropriate secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL High-Signal reagent (Thermo). The band intensity was quantified using the ImageJ software.