Xp rabbit mab

Nuciferine (purity > 98%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (China). RAW264.7 cells were obtained from the American Type Culture Collection (United States). LPS was obtained from Sigma (United States). IL-1β, IL-6, IL-10, and TNF-α ELISA kits were obtained from BioLegend (United States). Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb, NF-κB p65 (D14E12) XP^® Rabbit mAb, Phospho-IκBα (Ser32) (14D4) Rabbit mAb, IκBα (L35A5) Mouse mAb, and TLR4, β-actin were obtained from Cell Signaling Technology (Beverly, United States).

WB was performed following standard procedures [25 (link),41 (link)]. The gastric cancer tissues were homogenized using a grinding-tissue homogenizer. A RIPA lysis buffer (Solarbio, Beijing, China) was used to extract proteins from the cell lines or human tissues. Protein concentration was measured using the BCA method (Beyotime, Shanghai, China). Equal amounts of proteins were separated using 10–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with 5% skim milk, followed by incubation with primary antibodies (antibodies against STAT3, (124H6) Mouse mAb, cat# 9139T, Cell Signaling Technology, Danvers, MA, USA; Phospho-STAT3, (Tyr705) (D3A7) XP^® Rabbit mAb, cat#9145S, Cell Signaling Technology, Danvers, MA, USA; JAK2, cat#AF6022, Affinity; Phospho-JAK2, (Tyr1007), cat#AF3022, Affinity; N-cadherin, cat#E-AB-70061, Elabscience, Wuhan, China; Vimentin, cat#E-AB-70081, Elabscience) overnight at 4 °C. The samples were then incubated with a HRP-labeled secondary antibody (Elabscience, Wuhan, China) for 1 h at 25 °C. Finally, protein levels were detected using ECL reagents (GE Healthcare, Shanghai, China). Anti-GAPDH and anti-β-Actin antibodies (Elabscience, Wuhan, China) were used as internal controls. Original images of WB figures please see Supplementary File.

Total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with 120 V and then transferred onto nitrocellulose membranes. After the membranes were blocked with non-fat milk, the membranes were incubated with primary antibodies PARP (46D11) Rabbit mAb, IGFBP3 (D1U9C) Rabbit mAb and GAPDH (D16H11) XP® Rabbit mAb (catalog number: 9532; 25864; 5174, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Next, the membranes were incubated with anti-rabbit IgG, HRP-linked antibody (catalog number: 7074; Cell Signaling Technology). Finally, the blot was detected and analyzed by using Image-Pro Plus software (Mediacy, Inc., Rockville, MD, USA).

The K562 cells were cultured with compounds GP2, GP5, PT5 and imatinib at 10 µM concentrations. After 6 h of treatment at 37 °C, the cells were lysed in PBS-Laemmli sample buffer and then, immunoblot analysis was conducted using phospho-specific-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (1:1000) (Cell Signaling Technology, Danvers, MA, USA) or anti-β-actin clone AC-15 (Sigma-Aldrich, St. Louis, MO, USA). For immunoreactivity detection, the chemiluminescence method was performed as previously described [44 (link),45 (link)]. The band intensity was measured using the ImageJ software (NIH, Bethesda, MD, USA).