Horseradish peroxidase hrp conjugated goat anti rabbit igg antibody

After four weeks of feeding, six groups of mice (NCD, HFD, Fenofibrate, ON50, ON100, and ON200) were sacrificed to collect liver and adipose tissues. Murine liver and fat tissues were homogenized in 1 mL of RIPA buffer (10 mM Tris pH 7.8, 1 mM EDTA, 150 mM NaCl, phosphatase inhibitor, and protease inhibitor) using a homogenizer. Tissue lysate was collected and protein analysis was performed using Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Total protein (40 µg) was then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PVDF membrane transfer (Millipore, Bedford, MA, USA). Rabbit primary antibodies against carnitine palmitoyltransferase (CPT1α), UCP1α, adipocyte protein 2 (AP2), PPARγ, CCAAT/enhancer-binding protein α (c/EBPα) (Cell Signaling Technology, MA, USA), PPARα, peroxisomal acyl-coenzyme oxidase 1 (ACOX1), and SPHK2 (Abcam, Cambridge, MA, USA) were then used respectively to detect target proteins on PVDF membrane. These samples were normalized to mouse primary antibody against β-actin (Millipore, Cambridge, MA, USA). After incubation with secondary horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (Millipore, Bilerica, MA, USA), blots were developed with enhanced chemiluminescent substrate (Millipore, Bilerica, MA, USA). Bands were detected using a Chemiluminescence imaging equipment (Viber Lourmat, France).

8-week-old male C57BL/6 mice were purchased from the SLAC Laboratory (Shanghai, China) and were maintained in cages with constant temperature (21 ± 1 °C), relative humidity (60%), a strict 12 h/12 h light–dark cycle, and free access to water and food. The experimental protocol was approved by Institutional Animal Care and Use Committee of Henan Provincial People Hospital and carried out in accordance with the guidelines for the Care and Use of Laboratory Animals.
MiR-30e agomir, miR-30e mimics, and corresponding negative control miRNA were obtained from GenePharm (Shanghai, China). SuperScriptIII First-Strand Synthesis system, fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium, penicillin, streptomycin and lipofectamine 2000 were purchased from Invitrogen (CA, USA). Antibodies against α-syn, Nlrp3, ASC, Caspase-1 and β-actin were from Cell Signaling Technology (MA, USA). Tyrosine hydroxylase (TH) antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody, HRP-donkey anti-goat IgG antibody and the Enhanced Chemiluminesecence Kit were from Millipore (MA, USA). Nissl staining solution, diaminobenzidine (DAB) and RIPA buffer were purchased from Beyotime (Jiangsu, China). All chemicals and reagents unless otherwise indicated were obtained from Sigma (MO, USA).

Whole-cell extracts were prepared with 2 × SDS sample buffer (0.1 M Tris HCI, pH 6.8, 20% glycerol, 4% SDS, 10% β-Mercaptoethanol, 2% Bromophenol blue) and boiled for 10 min at 98 °C. Rabbit anti-γH2AX (S139) (9718), -H2AX (2595), -pATM (S1981) (13050), -pChk2 (T68) (2661), -Chk2 (2662), -pChk1 (S317), -PARP (9542), -caspase 7 (9492), -cleaved caspase 3 (D175) (9661), mouse anti-DNA-PK (3H6), and -caspase 8 (9746) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-ATM (1A1), -Chk1 (G-4), -CAD (F-11), goat anti-ATR (N-19), and rabbit anti-caspase 3 (H-277) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-pATR (T1989) was purchased from GeneTex (Irvine, CA, USA). Rabbit anti-pDNA-PK (S2056) was purchased from Abcam (Cambridge, UK). Mouse anti-Flag (M-2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-actin (20536) was purchased from Proteintech Group (Rosemont, IL, USA). Rabbit anti-PEDV N was generated previously in our lab [26 (link)]. The horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody and HRP-conjugated goat anti-mouse IgG antibody were purchased from MiliporeSigma (Merck Inc., Darmstadt, Germany).