Image quant las 4000

The protein was extracted from granulosa cells using RIPA lysis buffer (Beyotime, China) with proteinase and phosphatase inhibitors. The protein concentration was measured by a BCA protein assay kit (Beyotime, China). The samples were denatured at 95 °C for 10 min with 5× protein-loading buffer. The protein was separated by 10% SDS-PAGE and then was transferred onto 0.22-μm PVDF membranes (Millipore, USA). Next, the membranes were blocked with 5% skim milk for 1 h and were then incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were washed three times and incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:3000, Abcam, USA) for 1 h. Finally, the proteins were detected by enhanced chemiluminescence and were imaged by an Image Quant LAS 4000 mini biomolecular imager (Bio-Rad, USA).

The gel shift assay was done with 10 pmol of Cy5 3′ labeled dsDNA and DNA/RNA heteroduplex as a substrate and 10, 20, 50 or 100 pmol of AvaII dimer in 1 μl volume. The binding reaction was performed at 37°C for 30 min in the presence of bovine serum albumin (BSA) (1 mg/ml) and reaction buffer based on the NEBuffer 4 1× (20 mM Tris-acetate pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, H₂O) with 10 mM calcium chloride instead of magnesium acetate. Native gel electrophoresis was run in an 8% polyacrylamide gel in 89 mM TBE or TB buffer supplemented with 5 mM CaCl₂. After gel pre-run (10 V, 1 h) samples were loaded with 10 × loading dye solution (3 g Ficoll 400, Orange G dye, ddH₂O up to 10 ml) and run at 90 V. Fluorescently labeled oligonucleotides were visualized using ImageQuant LAS 4000 or ChemiDoc XRS+ (Bio-Rad). Protein was stained with Coomassie Brilliant Blue solution and destained with water.

Bacterial extracts, trypanosome whole cell protein lysates and purified proteins were separated on SDS-PAGE gels (10%–15%) and transferred by semi-dry (BioRad) blotting 45 min at 25V on PVDF membrane. After a 1 h blocking step in 5% Milk in PBS-0.2% Tween, the membranes were incubated with the primary antibodies diluted in blocking buffer–anti-TbSAXO (mAb25 mouse monoclonal, 1:1,000 dilution [80 (link)]); anti-FPC4 (rat, 1:500 dilution) and anti-myc (rabbit polyclonal (Santa Cruz), 1:500 dilution). After three washes in blocking buffer, the membranes were incubated with the secondary antibodies–anti-mouse antibody HRP-conjugated (Jackson, 1:10,000 dilution), anti-rat antibody HRP-conjugated (Jackson, 1:10,000 dilution) and anti-rabbit antibody HRP-conjugated (Sigma, 1:10,000 dilution)–and washed twice 10 min in blocking buffer and twice 5 min in PBS. Blots were revealed using the Clarity Western ECL Substrate kit (Bio-Rad) with the ImageQuant LAS4000.

Proteins isolated from the hippocampus and cerebral cortex tissue were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies (anti-MRP1 antibody 1:500, dilution, Abcam plc.; anti-p38 MAPK antibody, 1:1000 dilution, Cell Signaling Technology Inc.; anti-p-p38 antibody, 1:1000, dilution, Santa Cruz Biotechnology Inc.; and anti-GAPDH antibody, 1:30,000 dilution, Santa Cruz Biotechnology Inc.) at 4 °C overnight and subsequently horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Specific proteins were visualized with enhanced chemiluminescence detection reagent and determined by an Image Quant LAS4000 mini chemiluminescence imaging system with QUANTITY ONE software (Bio-Rad Laboratories, Hercules, CA, USA).

For western blot analysis, total proteins were extracted from radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, R0020). Protein was quantified by a bicinchoninic acid assay (BCA) protein assay kit (Beyotime, P0012S). Total protein was denatured by boiling for 10 min. Protein (40 µg) was separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, CA, USA) and transferred to a nitrocellulose filter membrane (Millipore, Massachusetts, USA). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies: rabbit polyclonal anti-endothelin A Receptor antibodies (Abcam, ab178454, 1:1000 in 5% skim milk), rabbit polyclonal anti-Endothelin B Receptor antibodies (Abcam, ab117529, 1:1000 in 5% skim milk), and rabbit polyclonal anti-GAPDH (Abcam, ab37168, 1:1000 in 5% skim milk) for 12 h to 16 h. Then, peroxidase-conjugated AffiniPure goat anti-rabbitIgG(H + L)(ZSGE-BIO, ZB-2301, 1:10,000 in 5% skim milk) was added for 2 h at room temperature and subsequently developed using an enhanced chemiluminescence reagent (Beyotime, P0018A). The signals emitted for chemiluminescence were detected using the Image Quant LAS 4000 (Bio-Rad, CA, USA, 00746947) and analysed with ImageJ software (NIH). The results were normalized to the corresponding densitometry signal of GAPDH.

Total protein (30 μg) was fractionated on 10% polyacrylamide gel and blotted onto a methanol-activated PVDF membrane. PVDF membrane was blocked overnight in blocking buffer (Supplementary Table 3) at 4°C. The blocking buffer was replaced with rabbit anti-FtsZ primary antibody (1:10000) solution for FtsZ or rabbit anti-RRF primary antibody (1:20000) solution for RRF. The membrane blots were washed and incubated with 1:10000 diluted anti-rabbit goat IgG (Sigma, Solna, Sweden) (Srivastava et al., 2016 (link)). The blots were washed and developed with X-ray film (Kodak) or chemiluminescence imager (ImageQuant LAS 4000) using Clarity Western ECL Substrate (Bio-Rad, Solna, Sweden).