Horseradish peroxidase conjugated goat anti mouse igg or goat anti rabbit igg

As previous describe [21 (link)], briefly a total of 100 porcine oocytes per group were lysed with 1 × sodium dodecyl sulfate sample buffer by heating at 98°C for 10 min. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked in 5% (w/v) skim milk for 1 h and then incubated at 4°C overnight with anti-SIRT1 (Cat #60303-1-Ig, Proteintech), anti-ubiquitin (Cat #ab19247, Abcam), or GAPDH (Cat #sc-365062, Santa Cruz), followed by incubation at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:20000; Santa Cruz Biotechnology). The membrane was detected using Pierce ECL substrate (Thermo Fisher Scientific). Blots were visualized using a CCD camera and UVISoft software (UVITEC, Cambridge, UK).

A total of 100 porcine blastocysts per group were lysed with 1 × sodium dodecyl sulphate (SDS) sample buffer by heating at 98°C for 10 minutes. Proteins were separated by SDS‐PAGE and transferred to polyvinylidene fluoride membranes. Next, the membranes were blocked in tris‐buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) non‐fat milk for 1 hour and then incubated at 4°C overnight with anti‐GSNOR (1:1000; Cat # 11051‐1‐AP, Proteintech) or β‐tubulin (1:1000; Cat # sc‐5274, Santa Cruz Biotechnology), followed by incubation at room temperature for 1 hour with horseradish peroxidase‐conjugated goat anti‐mouse IgG or goat anti‐rabbit IgG (1:1000; Santa Cruz Biotechnology). Blots were visualized using a CCD camera and UVISoft software (UVITEC Cambridge).

As previously report [23 (link)], a total of approximately 100 porcine oocytes per group were lysis with 10 μl RIPA buffer and 10 μl loading buffer, and then heated at 98 C for 10 min. Lysates were separated by 6–12% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked with 5% skim milk in TBST buffer for 1 h and then incubated with anti-PINK1, DRP1, LC3 or -GAPDH antibody at 4°C overnight. Subsequently, the membranes were washed with TBST buffer thrice and incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:20,000; Santa Cruz Biotechnology). Blots were visualized with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA) using a charge-coupled device camera and UviSoft software (Uvitec, Cambridge, United Kingdom).