Anti β actin primary antibody

2-Methoxyestradiol (2ME2) was purchased from Selleck (Houston, TX, USA). RPMI 1640, Ham's F-12K and fetal bovine serum were purchased from Gibco/BRL Life Technologies (Grand Island, NY, USA). Anti-HIF-1α primary antibodies were purchased from BioWorld (St Louis Park, MN, USA). Lamin B1 antibodies were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-β-actin primary antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Their respective horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Beyotime (Shanghai, China). PVDF membranes were purchased from Bio-Rad (Richmond, CA, USA). The enhanced chemiluminescence (ECL) agent was obtained from Thermo Fisher Scientific (Waltham, MA, USA). An Immunol Fluorescence Staining Kit with Alexa Fluor 555-Labeled Donkey Anti-Rabbit IgG and 2-(4-amidinophenyl)-6-indolecarbamidine (DAPI) were obtained from Beyotime (Shanghai, China). TRIzol was purchased from Invitrogen (Grand Island, NY, USA), and One Step PrimeScript RT-PCR Kit was obtained from TaKaRa (Dalian, Liaoning, China).

Cells and mice tissue were lysed in RIPA buffer with PMSF (Beyotime, Shanghai, China), and then protein was adjusted to the same level, added loading buffer and boiled at 100 °C for 5 min. A 50µg mass of protein lysates were loaded on 10% SDS-PAGE gels and transferred using PVDF membranes. After block, the membranes were incubated overnight with anti-SIRT3 (1:1000; Thermo, Waltham, MA, USA), anti-SIRT4 (1:1000; Thermo, Waltham, MA, USA), anti-SIRT5 (1:1000; Proteintech, Wuhan, Hubei, China) or anti-β-actin primary antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA), followed by incubation with appropriate secondary antibodies (1:10,000; Cell Signaling Technology, MA, USA) at 37 °C for 1h. The signal was visualized and captured with a ChemiDoc XRS+system (Bio-Rad, Hercules, CA, USA).

Western blot analyses were carried out as described previously [17 (link)]. The total protein of the placenta samples was extracted using a Tissue Protein Extraction Kit (CWBIO, Jiangsu, China), and the concentration of the extracted total protein was determined by a bicinchoninic acid assay (CWBIO, Jiangsu, China). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transmembrane experiments were carried out with the Mini-PROTEAN Tube Cell instrument (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies, including anti-FATP4 (ProteinTech, Rosemont, IL, USA), anti-CD36 (ProteinTech, Rosemont, IL, USA), anti-FABP5 (ProteinTech, Rosemont, IL, USA), and anti-β-actin primary antibodies (Cell Signaling Technology, Danvers, MA, USA). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz company, Hercules, CA, USA), and immunoreactive bands were visualized using the ECL kit (Beyotime, Shanghai, China) and documented with a chemiluminescence imaging system (UVP, Upland, CA, USA). The results of the densitometric analysis of bands were quantified by Image J 1.45 software (National Institutes of Health, Bethesda, MD, USA). To calculate the relative expression levels of the target proteins, all blots were standardized to β-actin.

The cells were harvested and lysed by RIPA buffer for 30 min at 4°C. 50 µg proteins were loaded into 15% SDS-PAGE for analysis. Rabbit polyclonal anti-HGF, anti-E-cadherin, anti-Vimentin or anti-β-Actin primary antibody (Cell Signaling USA, 1:1000 dilution) was added and the cells were incubated overnight at 4°C. Then, HRP (horseradish peroxidase) conjugate goat-anti-rabbit secondary antibody (Cell Signaling USA, 1:1000 dilution) was added and incubated for 4 h. The bound antibodies were detected using the ECL Plus Western Blotting Detection system (GE Healthcare). β-Actin was used as an internal control.

To regulate the expression level of ATG14 in CRC cells (HCT116 and SW480), pCMV plasmid was transfected using Lipofect8000 (Thermo Fisher, MA, USA) to induce gene overexpression. pCMV-ATG14-GFP vectors and cDNAs for human ATG14 were obtained from COBIOER (Nanjing, China) and Sino Biological Inc (Beijing, China), respectively. Then, the expression level of ATG14 in normal and ATG-overexpressed HCT116 and SW480 cell lines were determined by western blotting. Anti-ATG14 primary antibody (#5504) and anti-β-actin primary antibody (#3700) were obtained from Cell Signaling Technology Co. (MA, USA).