Chemidoc xr system

Lung tissues were homogenized in RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.) and the protein concentration was determined using the bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Total protein was separated by SDS-PAGE on 10% gels. In total, 30 µg protein was loaded in each lane. Proteins were then transferred to PVDF membranes (EMD Millipore). Membranes were blocked for 2 h at room temperature in 5% non-fat dry milk and incubated with a primary antibody anti-rat CCR7 (1:1,000; cat. no. ab32527; Abcam) or anti-OX62 (1:1,000; cat. no. bs-1274R; Bioss, Inc.) at 4°C overnight. Membranes were then incubated with the appropriate horseradish peroxidase-labeled anti-mouse [1:1,000; cat. no. GAM007; MultiSciences (Lianke) Biotech Co., Ltd.] or anti-rabbit IgG [1:1,000; cat. no. GAR0072; MultiSciences (Lianke) Biotech Co., Ltd.] secondary antibodies at room temperature for 50 min. Anti-β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology Inc.) or GAPDH (1:1,000; cat. no. sc-32233; Santa Cruz Biotechnology Inc.) was used as the loading control. Bands were visualized by autoradiography (ChemiDoc XR+System; Bio-Rad Laboratories, Inc.) and quantified by densitometry (ImageJ V1.46; National Institutes of Health). The results were normalized to GAPDH or to β-actin. All experiments were performed in triplicate.

After the end of exposure under conditions of simulated microgravity or hypergravity, oocytes from the experimental and control groups were collected from agar plates, lysed, and the membrane and cytoplasmic protein fractions were isolated according to [28 (link)]. For electrophoresis, the same amount of protein was applied to each well based on concentration measurements. After electrophoresis in SDS-polyacrylamide gel, the proteins were transferred onto nitrocellulose membranes. The loading control and transfer efficiency were assessed using Ponceau staining. The membranes were then washed in PBST, blocked in 4% skim milk powder, and stained with specific primary antibodies at dilutions recommended by the manufacturers (Table 1). HRP-linked horse and goat antibodies for chemiluminescent detection were used as secondary antibodies to detect rat IgG, mouse IgG, and rabbit IgG (#7077S, #7076S, and #7074S, accordingly, Cell Signaling Technology, Inc., Danvers, MA, USA) at a dilution of 1:10,000. The membranes were then treated with SuperSignal™ West Femto Maximum Sensitivity Substrate (#34,096, Thermo Scientific™, Waltham, MA, USA) at a dilution of 1:10. The protein bands were revealed using the ChemiDoc XR+ System and processed using Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA).

At the end of the culture, NHMs were washed twice with ice cold PBS and were lysed in RIPA buffer with the Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail (Thermo Scientific, Rockford, IL). Amounts of total protein were quantitated using BCA protein reagent (Thermo Scientifc, Rockford, IL). Total proteins (5 μg/lane) were denatured by heating at 95°C in Laemmli sample buffer (BioRad, Richmond, CA) for 5 min and loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels (BioRad, Richmond, CA). After electrophoresis, proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes and were immunoblotted with appropriate primary and secondary antibodies. Immunoblotted proteins were visualized using an ECL substrate (BioRad, Richmond, CA) and were detected and analyzed by ChemiDoc XR+ System and Image Lab software (BioRad, Richmond, CA).

Total cellular lipids were extracted by the method of Bligh and Dyer (Bligh and Dyer, 1959 (link)) and separated by thin layer chromatography. For phospholipid analysis, the plates were first developed in chloroform, methanol and 30% aqueous ammonium hydroxide (65:35:5, v/v), followed by chloroform, acetone, methanol, acetic acid and water (50:20:10:10:4, v/v) for the second direction. The separation of NBD-labeled lipids was obtained by one-dimensional thin layer chromatography using chloroform, ethanol, trimethylamine and water (30:35:35:7, v/v). NBD-labeled lipids were quantified on a ChemiDoc XR+ system with Image Lab software and Dylight 488 channel filter for Blue Epi illumination (Bio-Rad, Hercules, CA). Cellular lipids were visualized by Primuline staining (0.05% w/v in acetone:water, 8:2, v/v) using 365 nm UV light and quantitated by phosphate determination (Rouser et al., 1970 (link)). Phospholipids were identified by comparison with commercial phospholipid standards (Avanti Polar Lipids).

Human spermatozoa lysates were prepared from the resulting pellets. Pellets were resuspended in RIPA buffer and incubated for 15 min at 4 °C. The samples were then centrifuged (14,000× g for 20 min) and total protein concentration was quantified using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
The phosphorylation state of spermatozoa proteins was used as a biomarker to evaluate sperm capacitation [26 (link)]. Protein samples were diluted to a final concentration of 50 ng/µL and transferred to a PVDF membrane using a slot blot technique in a Hybri-slot manifold system (Biometra, Göttingen, Germany). A Ponceau S staining solution (MB19201, NZYTech, Lisboa, Portugal) was used for total protein normalization. The resulting membranes (n = 6 for each condition) were incubated overnight at 4 °C with a mouse anti-phosphotyrosine monoclonal antibody (1:1000, Clone 4G10, #05-321, Merck Millipore, Temecula, CA, USA). Membranes were then incubated with a goat anti-mouse IgG antibody (1:5000, AP308P, Sigma-Aldrich, St. Louis, MO, USA). Blots were visualized with Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and read using a Bio-Rad ChemiDoc XR system (Bio-Rad, Hercules, CA, USA). Densities from each band were quantified using Image Lab Software (Bio-Rad, Hercules, CA, USA).

For Western blot analysis, 20 μg of protein extract prepared as described previously [45 (link)] was separated by SDS-PAGE (10%). After electrophoretic transfer of the proteins, the nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) was stained with amido black to check for equal transfer across all lanes. The membrane was blocked for 1 h with 5% skimmed milk in phosphate-buffered saline (PBS)-Tween buffer (100 mM NaH₂PO₄-Na₂HPO₄, pH 7.4, 100 mM NaCl, 0.1% Tween) and incubated with a monoclonal RNase Y antibody diluted in PBS-Tween for at least 4 h. Signals were detected by ECL Chemiluminescence (BIO-RAD, Clarity^TM Western ECL) associated with a CCD camera (BIO-RAD ChemiDoc XR System+). When necessary, ECL-detected proteins were quantified using the ImageLab software version 6.0.1 (Bio-Rad, Hercules, CA, USA). The uncropped versions of the Western blots are shown in Supplementary Materials.

Cell lysis, protein separation and transfer onto polyvinylidene difluoride (PVDF) membranes were performed as previously described [26 (link)]. The membranes were incubated with the following primary monoclonal antibodies: anti-STAT3, anti-STAT3 (Ser727) (Cell Application, San-Diego, CA, USA), anti-AMPK, anti-phospho-AMPK (Thr172), anti-caspase 3, anti-DRP1 (Cell Signaling Technology, Danvers, MA, USA), and anti-phospho-DRP1 (Ser637) (Thermo Scientific, Waltham, MA, USA), followed by the incubation with HRP-conjugated goat anti-mouse IgG and anti-rabbit IgG (Sigma, St. Louis, MI, USA) secondary antibodies. HRP activity was developed by ChemiDocTM XR+ System (BioRad, Hercules, CA, USA) using a luminol-based enhanced chemiluminescence (ECL) HRP substrate Super-Signal West Dura (Thermo Scientific, Waltham, MA, USA). Densitometric analysis was performed using image processing Image Lab software version 5.2.1.