Rabbit anti α tubulin antibody

Protein was extracted from EndoC-βH1 cells in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with halt protease and phosphatase inhibitor Cocktail (Thermo Fisher Scientific). Protein samples were loaded onto NuPAGE 4–12% Bis–Tris Protein Gels (Thermo Fisher Scientific), resolved by electrophoresis and transferred onto nitrocellulose membranes. Membranes were incubated with the following primary antibodies: mouse monoclonal anti-β-actin antibody (Invitrogen, MA1-140; 1:20,000), rabbit monoclonal anti-phospho CHEK2 T68 antibody (Cell Signaling, 2197S; 1:1,000), mouse monoclonal anti-CHEK2 antibody (Cell Signaling, 3440T; 1:1,000), rabbit anti-eIF2α antibody (Cell Signaling, 5324; 1:1,000), rabbit anti-phospho eIF2α Ser51 antibody (Cell Signaling, 3597; 1:1,000), rabbit anti-α-tubulin antibody (Cell Signaling, 2144; 1:1,000), rabbit anti-phospho PLK1 T210 (Abcam, ab155095; 1:200) and mouse anti-PLK1 (Abcam, ab17056; 1:1,000). Primary antibodies were detected by fluorophore-conjugated secondary goat anti-rabbit (LI-COR IRDye 800CW, 926-32213; 1:15,000) and donkey anti-mouse (LI-COR IRDye 680RD, 926-32210; 1:15,000) using LI-COR Odyssey Imagers. Western blot images were analyzed with Image Studio software 5.2.5.

At 4 weeks after SM administration, the mice were superovulated via an intraperitoneal injection of 5 IU of pregnant mare serum gonadotropin (Prospec, Rehovot, Israel), followed by an injection of 5 IU of human chorionic gonadotropin (hCG; Prospec) 48 h later. Oocytes were collected at 18 h post-hCG injection into the preincubated human tubal fluid medium (Irvine Scientific, CA, USA). Oocytes were fixed with 4% paraformaldehyde (Biosesang) and permeabilised with 0.5% Triton X-100 (Sigma–Aldrich) for 10 min, followed by blocking with PBS containing 3% bovine serum albumin (GenDEPOT, TX, USA) and incubation with a rabbit anti-α-tubulin antibody (1:200; Cell Signaling Technology, MA, USA). Oocytes were mounted on slides using the VECTASHIELD antifade mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Peterborough, UK) to visualise the chromosomes and were observed under a fluorescent microscope (BX51; Olympus, Tokyo, Japan). Oocytes with well-organised bipolar spindles and tightly aligned chromosomes at the metaphase were scored as normal. The oocyte quality was evaluated by measuring morphometric parameters, including the areas of the whole oocyte, ooplasm, and PVS, using a computed image analysis system (Nikon Instruments).

Harvested cells were resuspended in 20 × 10⁻³m RIPA buffer (pH 7.4) (Merk Millipore, Vimodrone, MI, Italy) containing a cocktail of proteinase inhibitors (Calbiochem, Merck, Darmstadt, Germany) and treated by sonication (Microson XL‐2000, Minisonix, Farmingdale, NY, USA).
Aliquots of supernatants containing equal amounts of protein (30 µg) in Laemmli buffer were separated on Bolt Bis‐Tris Plus 4–12% precast polyacrylamide gels (Life Technologies, Monza, Italy). Fractionated proteins were transferred from the gel to a PVDF nitrocellulose membrane using an iBlot 2 system (Life Technologies, Monza, Italy). Blots were stained with Ponceau red to ensure equal loading and complete transfer of proteins, and then blocked for 1 h at room temperature with 5% milk in PBS containing 0.1% Tween. Subsequently, membranes were probed at 4 °C overnight with mouse anti‐uPAR antibody (1:500 Mon R4; ThermoFisher) or rabbit anti‐α‐tubulin antibody (1:1000 Cell Signaling) used to assess equal amounts of protein loaded in each lane. Antirabbit IgG (whole molecule)–Peroxidase antibody (Sigma, Cat#A0545) or antimouse IgG (whole molecule)–Peroxidase antibody (Sigma, Cat#A9044) were used as secondary antibodies; the enhanced chemiluminescence (ECL) procedure was employed for development.