Beyoecl moon kit

Briefly, extracted protein samples from cells were separated by SDS-PAGE, then transferred to nitrocellulose membrane and incubated with the primary antibodies. After incubation with horseradish-peroxidase-conjugated (HPR) secondary antibody for 2 h at room temperature, the protein expression was visualized with BeyoECL Moon Kit (Beyotime, Beijing, China) by Versa DosTM 4000 MP (Bio-Rad Laboratories, Munich, Germany).
Rabbit Anti-SOAT2 antibody (bs-5020R, Biosis, Beijing, China), Rabbit Anti-SREBP2 antibody (bs-2536R, Biosis, Beijing, China), LDL Receptor Rabbit Monoclonal Antibody (AF1438, Beyotime Biotechnology, Shanghai, China), PPAR-γ (I106) polyclonal antibody (BS1587, Bioworld Technology, Inc., MN, USA), CEBP Alpha Rabbit pAb (383901, ZENBIO, Chengdu, Sichuan Province, China), Anti-GAPDH [6C5]—Loading Control (ab8245, Abcam, Cambridge, UK).

Proteins were extracted from cells and quantified using BCA protein assay kit (Sangon Biotech, Shanghai, China). Proteins were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h before incubating with primary antibodies at 4 °C overnight. After 13–15 h, the membrane was washed with TBST 3 times and incubated with horseradish peroxidase-labeled secondary antibody (1:4000) at room temperature for 1 h. Finally, BeyoECL Moon Kit (Beyotime Biotechnology, Shanghai, China) was added for color development and image J software was used to analyze the absorbance value of the images.

Proteins were resolved by SDS-PAGE and electroblotted onto a PVDF membrane and probed with the indicated primary antibodies and then with secondary HRP-conjugated goat anti-mouse IgG (EarthOx, San Francisco, CA, USA). The signals were detected by a chemiluminescence reaction using the BeyoECL Moon Kit (Beyotime, Shanghai, China). Monoclonal anti-HA, anti-MYC and anti-GFP was used at dilution of 1:2000.

Freshly egressed parasites (1×10⁷ per sample) were purified by filtration to remove host cell debris, resuspended in 40 μl RIPA lysis buffer (Beyotime, China) and sonicated for 30 min in a water bath sonicator. Then, 10 μl DTT and 50 μl 2×SDS buffer were added to each sample and boiled for 10 min. Protein samples (10 μl each) were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Subsequently, the membranes were probed with indicated primary antibodies. Two different strategies were used to detect primary antibodies. For chemiluminescent detection, HRP-conjugated sheep anti-rabbit or sheep anti-mouse IgG (Epizyme, China) secondary antibodies (at the dilution of 1:2000) were used and the blots were developed by a BeyoECL Moon kit (Beyotime, China). For fluorescent detection, IRDye 800CW sheep anti-mouse IgG and IRDye 680RD sheep anti-rabbit IgG (LI-COR bioscience, USA) secondary antibodies (at the dilution of 1:5000) were used. The blots were scanned by a Tanon 5200 chemiluminescence imager (Tanon, China) and a Typhoon5 laser-scanner (GE Healthcare, USA).

Antibodies used in our experiments included anti-SHH, anti-Gli1, anti-snail1, and anti-GAPDH antibodies (Affinity Biosciences, China). PVDF membrane, BCA Protein Assay Kit, and BeyoECL Moon kit were acquired from Beyotime Biotechnology, China. Cyclopamine was purchased from Selleck Chemicals, USA. Experimental instruments included AU400 automatic biochemical analyzer (OLYMPUS, Japan), BX51T-PHD-J11 microscope (OLYMPUS Company, Japan), image acquisition system CMOS (OLYMPUS Company, Japan), and Image-Pro Plus (Media Cybernetics Company, USA), etc.

A549 cells (4 × 10⁵ cells/well) were incubated in 6-well plates for 24 h and treated with 0 and 160 μg/mL of C. oleifera bud EE for 48 h. Then, cell total proteins were isolated through RIPA lysis buffer, and protein concentrations were determined by a BCA protein assay kit (Solarbio, Beijing, China). Subsequently, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% milk in TBS-T (TBS, 0.1% Tween-20) for 1 h, and blotted with primary antibodies at overnight under 4°C. Next, membranes were washed three times with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h. Proteins were visualized using a BeyoECL moon kit (Beyotime, Shanghai, China), imaged with a ChemiDoc touch imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, United States), and quantified via Image Lab software.

Cell or tissue was lysed using RIPA lysis buffer (Bmassay), and Western blot was carried out based on published literature (Li et al., 2019 (link)). In brief, protein extracts were separated in 10% SDS polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking the membrane for 1 h, primary antibody was incubated overnight with gentle agitation. The primary antibodies for the target proteins included NPRA (Fabgennix), P21 (Proteintech), sirtuin 1 (SIRT1), PKG, AMPK and phosphorylated‐AMPK (p‐AMPK; Cell Signaling Technology). Subsequently, secondary antibody goat anti‐rabbit IgG or goat anti‐mouse IgG (ABclonal) was incubated at room temperature for 1 h. The blots were visualized employing a BeyoECL Moon kit (Beyotime). For probing multiple targets with the same membrane, stripping and re‐probing were performed. Briefly, the PVDF membrane was incubated with the stripping buffer containing 62.5 mM Tris–HCl (pH 6.8), 2% SDS and 100 mM β‐mercaptoethanol for 20 min at room temperature. After washing three times with TBST, the membrane was re‐incubated with primary and secondary antibodies.
The protein levels, normalized with GAPDH (Proteintech), were calculated by ImageJ (NIH).