β actin 2d4h5

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.

Isolated 1–3 × 10⁶ PBMCs and 0.5–1 × 10⁶ human vascular endothelial cells were pelleted and treated with homemade lysis buffer [48 (link)]. This is succeeded by measuring the protein concentration of the samples at 562 nm with a spectrometer (Tecan, Megelan, using Pierce^TM BCA Protein Assay Kit (Cat#23225, Thermo Fischer Scientific), where 20 µg of sample protein was subjected to SDS page electrophoresis and transferred to the PVDF membrane according to the standard laboratory procedure. We employed human primary antibodies for phospho-STAT-3 (Tyr 705) (D3A7), STAT-3 (D3Z2G), phospho-P65 (93H1), P65 (D14E12) (1:1000 or 1:2000), and cleaved caspase-1 (p-20) (Bally-1, Adipogen Life Sciences, San Diego, CA, USA) to detect the respective proteins. We used α-Tubulin (11H10) (Cell Signaling, Danvers, MA, USA) and/or β-actin (2D4H5) (Proteintech, Rosemont, IL, USA) as the loading protein and anti-rabbit IgG, HRP-linked antibody (Cat# 7074, Cell Signaling), and AP-conjugated anti-mouse IgG (S3721, Promega, Madison, WI, USA,) were used as secondary antibodies. As substrate, we used Pierce^TM ECL Western, Super Signal^TM West Femto, or BCIP/NBT (Promega). The subsequent development of bands was detected using an INTAS ECL ChemoStar Imager (INTAS science Imaging) and further densitometries were quantified using LabImage ID software.

Cells were lysed in RIPA buffer on ice for 30 min, shaken 3 times every 10 min, and then centrifuged at 12 000 rpm for 15 min. The proteins in the cell supernatant (protein lysates) were separated on 8% to 12% SDS‐PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at room temperature for 1 h, the PVDF membranes were incubated with the primary antibody at 4°C overnight. The following antibodies were used: β‐Actin (2D4H5, mouse, ProteinTech, Wuhan, China, 1:5000), OCLN (ab216327, rabbit, Abcam, Cambridge, MA, USA, 1:1000), p‐STAT3 (#9145, rabbit, CST, Danvers, MA, USA, 1:1000), STAT3 (#9139, rabbit, CST, Danvers, MA, USA, 1:1000) and HSP90 (3F11C1, mouse, ProteinTech, Wuhan, China, 1:5000). The corresponding secondary antibodies were as follows: HRP‐conjugated horse anti‐mouse (7076, CST, Danvers, Massachusetts, USA, 1:2000) and goat anti‐rabbit (7074, CST, Danvers, Massachusetts, USA, 1:2000) secondary antibodies. β‐Actin and HSP90 were used as the internal controls.