Image lab 4

Protein concentration was quantified using the Quantum Protein BCA Assay kit (Euroclone, Pero, Italy) in 80% confluent male and female AFCs after cell lysis. Western blotting was performed on 25 μg of solubilized protein as previously described [28 (link)]; protein expression was evaluated using the following primary antibodies: actin (1:1000; Sigma-Aldrich, Milano, Italy), ERα, ERβ (1:1000; Thermo Fisher Scientific, Rodano, Italy), LC3 (1:1000; MBL, Eppendorf Italia, Milano, Italy), p62 (1:1000), and LAMP1 (1:1000); these were obtained from Cell Signaling Technology (Milano, Italy) as primary antibodies.
After 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology, Milano, Italy), the binding was detected by chemiluminescence with the Bio-Rad ChemiDoc instrument (Bio-Rad, Milano, Italy). Band volume analysis was performed using the Image Lab 4.0 software (Bio-Rad, Milano, Italy). A pilot study (5 samples of each sex) suggested that in female and male AFCs, the expression of α-actin was very similar (827,366.79 ± 578,040.86 optical density (OD) and 839,428.71 ± 713,143.57 OD for females and males, respectively; p = 0.49). Therefore, it was used for the normalization of Western blot analysis.

Different concentrations of SV or PSD preparations and matching total brain homogenate were separated by SDS-PAGE on 4–12% polyacrylamide Bis–Tris gels (SV) (Invitrogen) or 7% SDS-PAGE/Tris–Glycine gels (PSD) and transferred to PVDF (SV) or nitrocellulose membranes (PSD) (Bio-Rad). The membranes were incubated with primary antibodies (Table 1) followed by secondary antibodies coupled to HRP (GE-Healthcare or Bio-Rad). Signals were visualized with SuperSignal West Pico enhanced chemiluminescent reagent (Pierce), Immobilon Western Chemiluminescent HRP Substrate (Millipore) or Clarity™ Western ECL substrate (Bio-Rad) and exposure to film (GE-Healthcare), LAS-3000 CCD camera (Fujifilm) or computer-assisted imaging (ChemiDoc system and Image lab 4.0 software; Bio-Rad).

Annealed oligonucleotides were loaded to 20% native polyacrylamide gel and electrophoresis was performed at 4 °C in Tris/Borate/EDTA (TBE) running buffer (90 mM Tris, pH 8.3, 90 mM boric acid and 5 mM EDTA). The gel were stained with SYBR^® Gold Nucleic Acid Gel Stain and visualized by Gel Dock XR + (Bio-Rad) and Image Lab 4.0 software (Bio-Rad). The fraction of monomolecular hairpin structures was evaluated based on the assumption that the efficiency of the staining of the base pairs in a hairpin was similar to that in a bimolecular duplex.

Protein extracts were resolved by 1-DE under reducing conditions and electrotransferred to nitrocellulose membranes in semi-dry conditions (Trans-Blot Turbo system; BioRad, Hercules, CA, USA). Serum amyloid P (SAP) detection was performed using a mouse monoclonal antibody against total SAP (ab27313, 1:200 dilution, abcam, Cambridge, UK). Band detection was performed using a chemiluminiscent substrate dye (Luminata Forte Western HRP Substrate, Merck Millipore, Billerica, MA, USA) and a molecular imager ChemiDoc XRS System, Universal Hood II (BioRad, Hercules, CA, USA). Band quantification was performed with Image Lab 4.0 software (BioRad Laboratories, Hercules, CA, USA). Protein load was normalized with total protein staining, as previously described [23 (link)].

Protein was extracted from total cell lysates by using RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) or from 48 h cell supernatant. Protein concentrations were measured with the Pierce BCA Protein Assay Kit (ThermoScientific). Twenty-five micrograms of protein was resolved by 1-DE under reducing conditions onto 10% SDS-PAGE gels and electrotransferred to nitrocellulose membranes. After blocking for non-specific binding with 5% of bovine serum albumin (BSA; MP Biomedical) or Blotto, the membranes were incubated with primary antibodies, including TF (4501-Sekisui Diagnostics), endostatin (ab109660-abcam), and VEGF (ab51745-abcam). Band detection was performed using a chemiluminiscent substrate dye (SuperSignal® West Dura Extended Duration Substrate, Thermo Scientific, Waltham, MA, USA) and a molecular imager ChemiDoc XRS System, Universal Hood II (BioRad, Hercules, CA, USA). Band quantification was performed with Image Lab 4.0 software (BioRad Laboratories, Hercules, CA, USA). Protein load was normalized with total protein staining, and normalization between different membranes was performed with a common pool in every gel, as previously described [24 (link)].

Protein concentration in cellular extracts was determined by the standard Bradford procedure (Bradford 1976 (link)). Samples of identical protein amount (30 µg) were separated by 10 % polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) according to Laemmli (1970 ), as a molecular mass marker served PageRuler™ Prestained Protein Ladder (Fermentas). This was followed by transfer to nitrocellulose membrane, using the procedure described elsewhere (Towbin et al. 1979 (link)). Monoclonal rabbit anti-gelsolin antibodies (Abcam, clone EPR1942) at dilution 1:5,000 were used to visualize gelsolin band on nitrocellulose. β actin and β tubulin recognized by monoclonal mouse anti-β actin (Sigma, clone AC-15) and anti-β tubulin (TUB 2.1) antibodies, were used as reference proteins. Secondary antibodies conjugated to horseradish peroxidase (HRP) were applied according to the manufacturer’s protocols (Cell Signaling). Immunoblots were developed using the Western blotting Luminol Reagent (Santa Cruz Biotechnology), photos of blots were taken with ChemiDoc™ MP System (Bio-Rad) and further analyzed with the help of ImageLab 4.0 software (Bio-Rad).