Hrp conjugated goat anti rabbit

Briefly, rBMSCs were seeded in 6-well plates at a density of 1 × 10⁵ cells/well and cultured with different concentrations of MHMs or S-MHMs for 48 h. For detection of HIF-1α, CoCl₂ was added to the medium at a final concentration of 100 μM. Equal amount of protein from each sample was separated on SDS-PAGE gels and then transferred to polyvinylidenedifluoride membranes (Millipore, MA, USA). After being blocked with milk for 3 h, the membranes were incubated with primary antibodies against HIF-1α (1:1000, rabbit anti rat; CST, USA), VEGF (1:1000, rabbit anti rat; Abcam, Australia) or β-actin (1:5000, goat anti rabbit; Abcam, Australia) overnight at 4 °C. Finally, the membranes were visualized with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Beyotime, China) using the ECL plus reagents (Solarbio, China) under a UVItec ALLIANCE 4.7 gel imaging system.

HUVECs were grown in DMEM supplemented with 10% FBS. Before processing the cells for western blotting, the culture medium was removed and the cells were washed three times with PBS. HUVECs were lysed with a lysis buffer (Beyotime, Haimen, China) on ice for 30 min. Cell lysates were centrifuged at 12,000 × g for 15 min at 4°C. Equal amounts of soluble protein were separated by 12% SDS-PAGE, and the protein bands were electro-transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk, followed by overnight incubation with the primary antibody. Secondary antibodies conjugated to horseradish peroxidase were used in the subsequent experiments. An enhanced chemiluminescence (ECL) detection kit was used to visualize the target proteins and internal control. Protein bands were obtained by autoradiography and analyzed via Quantity One 4.4 (Bio-Rad, Hercules, CA, USA). The specific antibodies used in this study were as follows: anti-IRE1α (ab48187, Abcam, Cambridge, England), anti-XBP1s (#12782, CST, Danvers, MA, USA), anti-CHOP (#2895, CST, Danvers, MA, USA), anti-BAX (#89477, CST, Danvers, MA, USA), anti-BCL2 (ab692, Abcam, Cambridge, England), anti-GAPDH (#5174, Danvers, MA, USA), HRP-conjugated goat anti-rabbit (Beyotime, Haimen, China, A0208) and HRP-conjugated goat anti-mouse (Beyotime, Haimen, China, A0216).

Immunodot blotting assay was performed as previously described (Cheng et al., 2014 (link)), with some modifications. Ten nanomolar DrRecJ, DrRecJΔC, DrRecJ-core, EcRecJ, DrHerA or DrNurA were spotted on a nitrocellulose (NC) membrane. Lysozyme was also spotted as negative control. The membranes were blocked in TBST containing 5% non-fat milk powder at 4°C for 2 h, followed by incubation in 1 μM purified DrRecJ, DrRecJΔC, DrRecJ-core, EcRecJ or DrHerA protein (100 mM NaCl, 20 mM Tris-HCl, [pH 7.5], 1 mM DTT, and 1 mM EDTA) at 4°C overnight. Membranes were washed by TBST (TBS containing 0.05% Tween 20) for three times, followed by incubation with the primary antibody, anti-RecJ or anti-HerA (prepared in our laboratory) with 1:1000 dilution, at 4°C for 4 h. Again, membranes were washed by TBST for three times and subsequently incubated with the secondary antibody HRP-conjugated goat anti-rabbit (Beyotime Biotechnology, China) with 1:10,000 dilution at 4°C for 4 h. Finally, the membranes were washed another three times with TBST and signals were detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo scientific).

Protein extracts were obtained from six ovaries and using the Cell Lysis Buffer for WB (Beyotime, P0013). The proteins were separated on 10% SDS-PAGE gel and transferred onto Immobilon-PSQ Transfer Membrane (Millipore MA, USA). After blocking, the membranes were incubated with the appropriate primary antibody (Supplementary Table S2) overnight at 4 °C. After washing three times in Tris-buffered saline and Tween 20 (TBST), the membranes were incubated at 37 °C for 2 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Beyotime, A0258) IgG or goat anti-mouse (Beyotime, A0216) IgG at 1 : 2000 dilution in TBST. Finally, the membranes were reacted with BeyoECL Plus Kit (Beyotime, P0018). β-Actin was used as housekeeping protein control.

For each sample, proteins were extracted from 3 to 4 ovaries using the RIPA lysis solution (Beyotime, P0013C) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. They were then transferred onto a polyvinylidene fluoride membrane (immobilon-P^SQ transfer membranes, Millipore, ISEQ00010, USA). After blocking in TBST buffer (TBS with Tween-20) containing 5% bovine serum albumin, the membrane was incubated in primary antibody (Table S2) overnight at 4 °C, washed and incubated with HRP-conjugated goat anti-rabbit (Beyotime, A0208) or anti-mouse IgG (Beyotime, A0216) diluted in TBST at room temperature for 1.5 h. Ultimately, the BeyoECL Plus Kit (Beyotime, A0018) was used for chemiluminescence; β-ACTIN was used as housekeeping protein as previous reported^{21 (link)}.

Proteins were extracted from gonads with RIPA lysis buffer (Beyotime, P0013C, China) and then boiled for 5 min as previously described [50 (link), 51 (link)]. Primary antibodies, including rabbit anti-TCFL5 (1:400, Abcam, ab188075, USA), mouse anti-SYCP3 (1:1000, Abcam, ab97672, USA), rabbit anti-STRA8 (1:1000, Abcam, ab49405, USA), and rabbit anti-GAPDH (1:1000, Affinity, AF7201, China), were used. Secondary antibodies were HRP-conjugated goat anti-rabbit (1:1000, Beyotime, A0258, China) or mouse IgG (1:1000, Beyotime, A0216, China).