Quantity one software

Protein extraction and Western blot analysis were performed as described previously (15 (link), 25 (link), 39 (link)). Briefly, 50 µg proteins were resolved in 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 1% BSA in Tris-buffered saline with 0.1% Tween for 2 h at room temperature, membranes were incubated overnight at 4°C with the specific antibody (dilution 1:1000). Subsequently, blots were re-probed with the respective total antibodies (1:1000) or GAPDH (1:1500) for normalization. Immunoreactive proteins were visualized using horseradish peroxidase-conjugated goat anti-mouse (1:4000) and goat anti-rabbit (1:10000) secondary antibodies by enhanced chemiluminescence substrate (ECL, Bio-Rad, Milan, Italy) using ChemiDoc XRS (Bio-Rad, Milan, Italy); densitometric analysis was performed with Quantity One software (Bio-Rad, Milan, Italy). Each experiment was performed in triplicate. Densitometric analysis was carried out with Quantity One software (Bio-Rad).

Detergent extracts of the HCMEC/D3 cells and hCMEC/D3-MDR1-EGFP cells were prepared as described before21 (link) and subjected to Western blot analysis using anti-PGP 1:200 (Signet Laboratories, Dedham, MA, USA), anti-acetyl-histone-H4 1:200 (pan Lys 5, 8, 12, Merck Chemicals GmbH, Schwalbach, Deutschland) and anti-actin 1:1000 (Sigma-Aldrich) as primary antibiodies. The secondary antibodies utilized anti-mouse-HRP 1:1000 and anti-rabbit-HRP 1:1000 (Dako, Hamburg, Germany). Proteins were visualized by enhanced chemiluminescence using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc system (Bio-Rad, Munich, Germany) with QuantityOne software (Bio-Rad) according to the manufacturer’s instructions. The indicated protein signals were quantified densitometrically with QuantityOne software (Bio-Rad) and calculated by normalization to the reference signals of actin with GraphPad Prism software (GraphPad, San Diego, CA, USA).

More than 500 pollen grains were photographed using SEM, and the aborted pollen grains were numbered. The relative fluorescence fold change of DiOC₂ was quantified by measuring 80 pollen grains from 6 differential anther sections using Quantity One software (Bio-Rad, United States). The relative fold change of pollen coat concentration was quantified by measuring 60 pollen grains from 20 differential TEM sections using Quantity One software (Bio-Rad, United States). One-way ANOVA or Student’s t-test was used to evaluate the statistical significance between different genotypes. Different lowercase letters above the brackets represent statistically significant differences.

The tissue samples were collected from humans and mice for Western blot analysis using a previously described protocol^{4 (link),25 (link)}. Briefly, total proteins were extracted according to the manufacturer’s protocol (Keygen Biotech, Nanjing, China). SDS-PAGE gels (5% stacking gel; 10% separating gel) were used to separate total protein lysates (40 μg per lane) and electrophoretically transferred to polyvinylidene fluoride membranes (PVDF) (Millipore Corporation, USA). PVDF membranes were incubated with 5% nonfat milk for 1 h at room temperature (RT) to prevent non-specific binding. Later, the membranes were incubated with rabbit anti-GHRH (1:500, catalog No: ab187512, Abcam, Cambridge, MA, USA) and anti-GAPDH (1:3,000, Proteintech, Wuhan, China) antibodies overnight at 4 °C. The secondary antibody (peroxidase-conjugated goat anti-rabbit IgG (1:3,000, Proteintech, Wuhan, China) was incubated with the PVDF membranes for 1 h at RT on the next day. Enhanced chemiluminescence (ECL) reagent (Thermo, Marina, CA, USA) and a Fusion FX5 image analysis system (Vilber Lourmat, Marne-la-Vallée, France) were used to visualize the bands. Finally, Quantity One software (Bio-Rad, CA, USA) was used to measure the resulting optical density (OD) values, which were normalized to GAPDH expression.

The total protein of cells or tissues were extracted and prepared for western blot. The BCA Kit (Sigma-Aldrich, USA) was utilized to quantify the protein concentration. By the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, proteins were separated into different bands according to the molecular weight. Subsequently, proteins in different bands were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Then the membranes were blocked with 5% nonfat milk for 1 h. Primary antibodies (anti-TGF-α, LC3B, p62, cleaved caspase-3, cleaved PARP, β-actin, with different concentration 1:500, 1:3000, 1:1000, 1:500, 1:1000, 1:500, respectivly, BOSTER, Wuhan, China) were incubated at 4˚C overnight. Next day, after membranes were washed with TBST buffer for three times, the corresponding secondary antibodies labelled with the HRP (BOSTER, Wuhan, China) were incubated at room temperature for 2 h. Finally, the signaling was visualized using enhanced chemiluminescence and the relative concentrations were evaluated by Quantity One software (Bio-Rad, Hercules, CA, USA).