Horseradish peroxidase hrp conjugated secondary antibody

Perfluorooctane sulphonate (PFOS; potassium salt, purity ≥ 98%, Sigma-Aldrich, Shanghai, China) was dissolved in dimethyl sulphoxide (DMSO; Sigma-Aldrich, Shanghai, China) and stored at 20°C. N-acetyl-cysteine (NAC), 3-methyladenine (3-MA), and chloroquine (CQ) were purchased from Sigma-Aldrich (St. Louis, MO, USA; A7250-10G, M9281-100MG, and C6628-25G, respectively). Methyl thiazolyl tetrazolium (MTT) was purchased from Amresco (Solon, OH, USA, 0793-5g). A ROS assay kit was purchased from Beyotime Biotech (Nanjing, China, S0033). An AV-FITC/PI kit was purchased from KeyGen Biotech (Nanjing, China, #KGA108). Monodansyl cadaverine (MDC) and rhodamine 123 (Rh123) were purchased from Beyotime Biotech (P6659-20 μg and C2007, respectively). A BCA kit was purchased from Beyotime (Shanghai, China, P0012S). Anti-p62 was purchased from Proteintech (Shanghai, China, 55274-1-AP). Anti-LC3 was purchased from Sigma-Aldrich (Shanghai, China, L8918-200 μL). Anti-Bcl-2 and anti-Bax were purchased from CST (Shanghai, China, #3498 and #5023, respectively). Anticleaved-caspase-3 and β-actin were purchased from Santa Cruz, Paso Robles (CA, USA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies and the SuperSignal West Pico Kit were purchased from Thermo (Waltham, MA, USA).

After treatment, cell pellets were collected, resuspended with 1x RIPA lysis buffer [50 mM Tris-HCl at pH = 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na₃VO₄, 1 mM dithiothreitol (DTT), 1 Mm phenylmethyl sulfonyl fluoride (PMSF)], and incubated on ice for 1 h. Cell lysates were collected by centrifugation at 13,000 rpm for 15 min, and protein concentration was measured using a BCA protein assay kit (TAKARA, Seoul, Republic of Korea). An equal amount of cell lysates was resolved by SDS-PAGE and transferred to PVDF membranes (Merck-Millipore Korea, Daejeon, Republic of Korea). The membranes were incubated in blocking buffer (5% skim milk in 1x PBS with 0.1% Tween-20, 1x PBST) for 1 h and hybridized with appropriated primary antibodies in 1x PBS overnight at 4 °C. After washing three times with 1x PBST for 30 min, the membranes were hybridized with horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo-Fischer Scientific, Waltham, MA, USA) for 1 h at 4 °C and washed three times with 1x PBST for 30 min. The membranes were visualized by using enhanced chemiluminescence (ECL) detection system. Western blot was conducted in triplicates, and the images of the films were subjected to densitometry analysis.

Chemokine mixtures or the OHD_4–12 were electrophoretically separated in non-denaturing or denaturing conditions using 16.5% polyacrylamide gel and then transferred to 0.2 μm nitrocellulose membrane using a semi-transfer apparatus (Biorad). Membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk at 4 °C and incubated with the primary anti-CXCL4 and anti-CXCL12 antibodies (MAB7952 and MAB310, respectively, R&D systems) overnight. The membranes were then incubated with species-specific horseradish-peroxidase (HRP)-conjugated secondary antibodies (#31450, ThermoFisher). Following the incubation with ECL substrate (BioRad), the presence of specific proteins was determined based on chemiluminescence detected using a ChemiDoc Imaging System (BioRad). For co-IP analysis, chemokine mixtures were incubated with magnetic microbeads pre-coated with mouse anti-CXCL4 or anti-CXCL12 antibodies (R&D Systems) at 4 °C for 2 h. After incubation, microbeads were magnetically bound and washed to remove the unbound fractions. After elution, the Western blot analysis of the immune-precipitated samples was performed using goat anti-CXCL12 monoclonal antibodies (R&D Systems).

The SRC inhibitors (dasatinib, bosutinib, and saracatinib) were purchased from Selleckchem (Houston, TX, USA). All chemicals were dissolved in dimethyl sulfoxide (DMSO) and aliquots were stored at −80 °C. Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA).