Gel transfer system

Gelatinolytic activity within oviduct tissue homogenates was determined using transfer zymography as described previously by Pan et al. (2011 (link)). Briefly, samples (50 μg of total protein) containing proteolytic enzymes were first resolved in nonreducing SDS–PAGE on a 10 % gel without substrate. The proteins in the resolving gel were then electrophoretically transferred, using Invitrogen gel transfer system, to a previously prepared gel with a copolymerized gelatin (0.5 mg/ml). The receiving gel was then washed 3 times for 15 min in 2.5 % (v/v) Triton X-100 with gentle shaking to remove SDS and incubated at 37 °C overnight in developing buffer (50 mM Tris-HCl, 10 mM CaCl₂, 200 mM NaCl, 0.05 % NaN₃, pH 7.5). Subsequently, the gel was stained with 0.5 % (w/v) Coomassie Brilliant Blue R-250 and destained in distilled water until clear bands indicating gelatinolytic activity became visible. An extended-range protein ladder was used for molecular weight estimation of the bands. Negative control consisted of 12 mM 1,10-phenanthroline or 10 mM EDTA (chelates Zn²⁺ and Ca²⁺) added to renaturing and developing buffer before gel incubation.

To prepare cell lysates, cells were collected at 70–80% confluence 48 h post-transfection. Protein concentrations were determined, and a loading buffer was prepared according to the Solabank BCA Protein Concentration Assay Kit (Beijing, China). The prepared loading buffer was applied to individual lanes on a 4–15% gradient polyacrylamide gel (Mini-PROTEAN TGX™, Bio-Rad). Blotting was executed using a gel transfer system (Invitrogen) to transfer proteins onto PVDF membranes, which were then immersed in TBST (TBS containing 5% skim milk powder and 0.1% Tween) for 2 h. Following this, the blotted membranes were incubated with primary antibodies at a concentration of 1:1000 overnight at 4 °C. After washing the membrane three times, it was incubated with rabbit-specific secondary antibody (1:3000) for 2 h at room temperature. To develop the blotted membrane, the membrane was washed thrice with the ELC kit (New Cell & Molecular Biotech Co., Ltd. SuZhou, China), and the grayscale values of the target bands were assessed utilising Image J. GAPDH expression level was employed to normalise the target proteins. All antibodies were sourced from Abcam, including CD274 (ab205921), PI3K (ab302958), p-PI3K (#4228), p-AKT (ab8805), AKT (ab8805), and GAPDH (ab8245).