o, IL, USA) in PBS (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) containing 20 mM imidazole, based on the manufacturer’s recommendations. PBS containing 500 mM imidazole was utilized for the final elution step. Finally, the imidazole concentration was reduced to less than one hundred mM with an Amicon PLBC Ultracel-3 kDa membrane filter unit (Millipore), in PBS (pH 7) and the protein fraction was kept frozen at -70 C until further use. 4.5. Sea Bass Amhr2-Directed Antibodies The rabbit antisera were made on demand by Agrisera AB (V n , Sweden). AntiAmhr2 was raised against a synthetic peptide corresponding to amino acids I67NGQPQVDLLAC78 of sea bass Amhr2, located inside the extracellular domain. The antibody was affinity-purified against the synthetic peptide applied for the immunization protocol and its titer was tested by enzyme-linked immunosorbent assay. 4.6. Western Blot For immunoblotting, diverse quantities of recombinant sea bass Amh, and protein extracts from CHO/HEK293/COS-7 cells transfected with Amhr2 or pcDNA3, previtellogenic ovaries and follicular cells had been mixed with Laemmli sample buffer and distilled water, denatured at 95 C for 5 min and subjected to 125 SDS-PAGE. Right after electrophoresis, the proteins had been transferred onto previously activated PVDF membranes (Immobilon P, Millipore, Burlington, MA, USA) utilizing the Trans-Blot TurboTM Blotting Method (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for 1 h at room temperature in Tris-buffered saline with Tween 20 (TBST; 20 mM Tris, 140 mM NaCl, 0.1 Tween, pH 7.six) and blotting-grade blocker (five non-fat milk powder; Bio-Rad). Then, membranes have been incubated with all the sea bass anti-C Amh antibody (1 /mL, [30]) or sea bass anti-Amhr2 antibody (two /mL) overnight at 4 C. Bound antibodies have been detected with 1:25,000-diluted goat anti-rabbit IgG (Sigma-Aldrich, Inc., Saint Louis, MO, USA) coupled to horseradish peroxidase (HRP), and proteins were visualized by utilizing enhanced chemiluminescence (PierceTM ECL Plus Western Blotting Substrate) in the Amersham Imager 600 (GE Healthcare Bio-Sciences, CDK1 Activator medchemexpress Chicago, IL, USA). Quantification of band density was carried out utilizing the ImageQuantTM TL software (GE Healthcare Bio-Sciences, Chicago, IL, USA). A recognized concentration of sea bass AmhC created in CHO cells [30] CB1 Activator custom synthesis served as a typical curve for the semi quantification of P. pastoris sea bass AmhC concentration. four.7. Cell Culture, Transfection and Luciferase Assay African green monkey kidney fibroblast-like (COS-7) cells have been made use of to express the sea bass Amhr2 protein as previously described [30]. Cells have been seeded onto 24 well plates ( 1.5 105 cells per nicely) in Dulbecco modified Eagle medium (DMEM) GlutaMAX (LifeInt. J. Mol. Sci. 2021, 22,14 ofTechnologies, Inc., Life TechnologiesTM Ltd., Paisley, Scotland, UK) supplemented with ten v/v heat-inactivated FBS, and one hundred U/mL of penicillin and streptomycin, at 37 C within a 5 CO2 incubator. Cells were grown to 750 confluence and co-transfected employing FuGENEHD Transfection Reagent (Promega) together with the following plasmids: (i) the BRE-Luc reporter plasmid (one hundred ng), which has many optimized BMP-responsive elements driving the expression from the firefly luciferase gene [70], (ii) the pcDNA3-Amhr2 expression plasmid (415 ng) [30], and (iii) the pRL-TK reporter plasmid (Promega, Corp., Madison, WI, USA) (20 ng), which constitutively expresses the Renilla luciferase gene, to normalize transfection. Different amounts on the empty
