Anti iκb

C57BL/6 mice (female, 6–8 weeks old) were purchased from the Laboratory Animal Center of Soochow University. During the experimental period, the mice were housed in ventilated cages in a specific pathogen‐free facility at room temperature (22–24°C) and kept on a 12‐h light/dark cycle, with water ad libitum and standard chow. P7C3 was obtained from MedChemExpress (Monmouth Junction, NJ, USA) and stored in dimethyl sulfoxide (DMSO) at −20°C. Recombinant mouse macrophage‐colony stimulating factor (M‐CSF) and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). Soluble, recombinant human M‐CSF, RANKL and tumor growth factor β (TGF‐β) were obtained from PeproTech (London, UK). Anti‐Akt, anti‐phospho‐Akt, anti‐ERK, anti‐phospho‐ERK, anti‐p38, anti‐phospho‐p38, anti‐JNK, anti‐phospho‐JNK, anti‐NFATc1, anti‐CTSK, anti‐MMP‐9, and anti‐GAPDH antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti‐TRAF‐6, anti‐IκB, anti‐p65‐NF‐κB, and anti‐phospho‐p65‐NF‐κB antibodies were purchased from Abcam (Cambridge, MA, USA). Alpha‐minimum essential medium (α‐MEM), fetal bovine serum (FBS), streptomycin, and penicillin were obtained from Gibco BRL (Grand Island, NY, USA).

Protein preparations from the indicated cells were obtained by lysing samples in 50 mmol/L of TRIS (pH 7.5), 100 mmol/L of NaCl, 1% NP40, 0.1% Triton, 2 mmol/L of EDTA, 10 Ag/mL of aprotinin, and 100 Ag/mL of phenylmethylsulfonyl-fluoride. Prestained molecular weight standards were from Bio-Rad (Milan). Proteins separated on the polyacrylamide gels were blotted on a PVDF membrane. The membrane was stained with Ponceau S (Sigma) to enable us to evaluate the success of transfer, and to locate the molecular weight markers. Free protein binding sites on the PVDF membrane were blocked with nonfat dry milk and a Tween 20/TBS solution. The membranes were washed and stained with specific primary antibodies and with secondary antisera, and then conjugated with horseradish peroxidase (Sigma-Aldrich) diluted 1:2,000. The anti-E-cadherin, anti-N-cadherin, anti-CXCR2, anti-phospho-P38, anti-P38, anti-IκB, anti-NF-κB, anti-slug, anti-twist1, anti-ZEB1, anti-Snail and anti-SIP1 antibodies were purchased from Abcam, and the anti-GRO-α antibody was from Santa Cruz. The luminescent signal was visualized with the ECL Western blotting detection reagent kit (Amersham) and quantified by scanning with a Discover Pharmacia scanner equipped with a Sun Spark Classic Workstation.