P p38 d3f9

Ulinastatin was purchased from Techpool (Guangzhou, China), and dissolved in normal saline and serum-free α-MEM basic medium for using in vivo and in vitro, respectively. Recombinant soluble mouse RANKL and macrophage-colony stimulating factor (M-CSF) were obtained from Peprotech (Rocky Hill, CT, United States). Rabbit antibodies against p38(D13E1, #8690), p-p38(D3F9, #4511), ERK(137F5, #4695), p-ERK(D13.14.4E, #4370), JNK (#9252), p-JNK(81E11, #4668), p65(D14E12, #8242), p-p65(93H1, #3033), IκBα(L35A5, #4814), and p-IκBα(14D4, #2859) were acquired from Cell Signaling Technology (Boston, MA, United States). Mouse antibody against uPAR (ab103791) was obtained from Abcam (Cambridge, MA, United States). Mouse antibody against GAPDH (A00227-1, P04406) was obtained from Boster (Wuhan, China).

J774 samples were sonicated and solubilized in Laemmli buffer. The proteins were separated using SDS-Page Electrophoresis and transferred to the nitrocellulose membrane. The membrane was blocked in 5% non-fat dry milk in TBS (10mM Tris, 150mM NaCl; pH 8.0) for 30 min. Specific primary antibodies were diluted in 1% non-fat dry milk or BSA in TBS. The nitrocellulose membrane was incubated with the following primary antibodies against extracellular signal-regulated kinase (ERK)1/2 (137F5; Cell Signaling, Danvers, MA, USA), p-ERK1/2 (197G2; Cell Signaling), p38 (sc-535; Santa Cruz, Dalas, TX, USA) or p-p38 (D3F9; Cell Signaling), and gently rocked at 4 °C overnight, followed by three 10 min washes in TBS containing 0,3% Tween-20. β-Actin (sc-47778, Santa Cruz) was used as a protein-loading control. Subsequently, the membrane was incubated with appropriate HRP-conjugated secondary antibodies for 1 h at RT and followed by three 10 min washes as mentioned above. Protein bands were visualized using enhanced chemiluminescence according to the manufacturer’s instructions and developed using the ChemiDoc Imaging System (BioRad; USA). The developed images were analyzed using ImageLab software.