Secondary antibodies conjugated with horseradish peroxidase

Western blotting was performed using tumors harvested 4 hours post TKI dosing on the last day of drug administration. Dissected frozen tumors were homogenized using TissueLyser II (Qiagen, Hilden, Germany) in T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL, USA) with a protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Homogenates were centrifuged and analyzed using western blotting. For the KIT signaling pathway analysis, antibodies against phosphorylated p-KIT^Y719, p-KIT^Y703, KIT, p-p85^Y607, p85, p-AKT, AKT, p-mechanistic target of rapamycin (mTOR)^S2448, p-extracellular signal-regulated kinase (ERK), ERK p-S6^S235/236, p-S6^S240/244, S6, p-signal transducer and activator of transcription 3 (STAT3), and STAT3 were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody against actin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

BV-2 cells were ultrasonically fragmented and lysed with radioimmunoprecipitation assay buffer (RIPA, Beyotime, Shanghai, China) on ice for 30 min. The extracted protein concentration was determined using the BCA protein assay kit (Solarbio, Beijing, China). Proteins were separated via SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% BSA for 2 h, the membranes were incubated with the primary antibodies (TNF-α, Cat#17590, Proteintech; IL-6, Cat#66146, Proteintech; IL-1β, Cat#12507, Cell Signaling Technology; iNOS, Cat#AF7281, Beyotime; TLR4, Cat#66350, Proteintech; α-Tubulin, Cat#T9026, MilliporeSigma) at ≥ 1/1000 dilutions at 4°C overnight, and then incubated with secondary antibodies conjugated with horseradish peroxidase (Cat#111-035-003, Cat#115-035-003, Jackson ImmunoResearch) at 1/5000 dilution for 1 h at room temperature. Finally, the protein bands were detected using BeyoECL Star (Beyotime, Shanghai, China) and quantified using the Alphaview SA software for Fluor Chem FC3 (ProteinSimple, San Jose, CA, USA).

The prostate cancer cell lines LNCaP and PC3 and HCT116 colon cancer cells were obtained from Georgetown University (GU) Lombardi Comprehensive Cancer Center (LCCC) cell culture repository and cultured in DMEM (Invitrogen, Carlsbad, CA) medium supplemented with 10% fetal bovine serum (FBS), 2mM-glutamine, 25ug/ml of gentamicin (Invitrogen) and incubated at 37^◦C with 5% CO₂. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (Allendale, NJ) and cultured as described [49 (link)]. The following primary antibodies: SIRT1, LC3B-I/II, and GAPDH antibodies were purchased from (Cell Signaling Technology Inc., Danvers, MA). Secondary antibodies conjugated with horseradish peroxidase were from Jackson ImmunoResearch (West Grove, PA).