P iκb α 14d4

Ginkgo biloba L. leaf extract injection (GBE) was purchased from Youcare Pharmaceutical Group (Beijing, China, Approval No. H20070226, Supporting Information 1). Its initial concentration is 3.5 mg/ml and prepared in aqueous solvent with excipients such as sorbitol, ethanol, sodium hydroxide. Lipopolysaccharide (LPS, #L2880) were obtained from sigma, and prepared in DMEM medium at concentration of 10 mg/ml. Primary antibodies for COX-2 (#4842, 1:1000), IκB-α (L35A5) (#4814, 1:1000), p-IκB-α (14D4) (#2859, 1:1000), NF-κB p65 (D14E12) (#8242, 1:1000) and p-p65 (93H1) (#3033, 1:1000), NF-κB p50 (D4P4D) (#13586, 1:1000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, United States). Primary antibodies for TNF-α (7B8A11) (#60291-1-Ig, 1:1000), IL-6 (#21865-1-AP, 1:1000), GAPDH (1E6D9) (#60004-1-Ig, 1:1000), β-actin (2D4H5) (#66009-1-Ig, 1:1000) and Lamin B1 (#12987-1-AP, 1:1000) were obtained from Proteintech Group Inc. (Rosemount, IL, United States). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Laboratories.

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).

Cellular protein was extracted by RIPA Lysis Buffer (Cell Signaling) supplemented with a protease inhibitor cocktail (Applygen). Proteins concentration was determined using the Pierce™ BCA Protein Assay kit (Thermo Scientific). Proteins were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membrane (Schleicher & Schuell). The membranes were blocked with 5% milk and then incubated with antibodies against p-Src (Y416) (2101) (1:1000; Cell Signaling), total-Src (36D10) (1:1000; Cell Signaling), p-STAT3(D3A7) (1:1000; Cell Signaling), total-STAT3 (9132) (1:1000; Cell Signaling), NF-κB(p65) (D14A12) (1:1000; Cell Signaling), Arg-1 (sc-271430) (1:1000; Santa Cruz), or iNOS (sc-650) (1:1000; Santa Cruz), total IκBα (44D4) (1:1000; Cell Signaling), p-IκBα (14D4) (1:1000; Cell Signaling). Antibody incubation was performed overnight at 4°C, membranes were sufficiently washed and then incubated with appropriate HRP-conjugated secondary Antibodys for 1 h at room temperature. Proteins were detected by ECL detection reagent. Expressions of proteins were normalized to β-actin.