Protein a g beads

Co-IP assay was conducted with the adoption of Co-IP kit (ACE Biotechnology, Nanjing, China). In short, the acquired cell lysates by centrifugation (14,000 rpm, 4 °C, 15 min) were treated by 2 μg of PLXNC1 (cat. no. sc-390216; Santa Cruz Biotechnology) or GRP78 antibodies (cat. no. sc-13539; Santa Cruz Biotechnology) or goat anti-mouse IgG (cat. no. ab6789; Abcam), followed by mixture with protein A/G beads (Shanghai Yeasen Biotechnology Co., Ltd.). Analysis of PLXNC1 and GRP78 enrichment was implemented by western blotting.

The 30 μL protein- A/G beads (Cat# 36403ES03, Yeasen Co., China) were washed three times with 300 μL immunoprecipitation (IP) buffer (1 mmol/L EGTA, 20 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 10 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 0.05% Triton X-100, 0.05% Nonidet P-40) with 1:500 phosphatase inhibitor and 1:100 protease inhibitor, centrifuged at 200 g for 1 minute at 4 °C. For IgG-protein-A/G coupling, 5 μg rabbit anti-fidgetin antibody or control rabbit IgG were coupled to cleaned 30 μL protein-A/G beads in 250 μL IP buffer and rotated on a flip shaker for 4 hours at 4 °C. Next, the IgG-coupled protein A/G was washed three times (10 minutes each) and centrifuged at 200 g for 1 minute.
At the same time, for oocyte lysate precleaning, 600 oocytes were lysed in 250 μL IP buffer by ultrasound, then 30 μL cleaned protein-A/G beads were added to oocyte lysis solution and rotated on a flip shaker for 4 hours at 4 °C. Next the mixture was centrifuged at 200 g for 1 minute at 4 °C and the oocyte lysate was kept. Then the immunoprecipitation was performed by adding the IgG-coupled protein-A/G beads into the precleaned oocyte lysate and rotated on a flip shaker overnight at 4 °C. After that, the immunocomplex was washed three times in 250 µL IP buffer and boiled in protein sample buffer (Cat# BD0034-3, Bioworld Inc., USA) for 5 minutes, followed by SDS-PAGE.

The testis samples were dissected immediately from adult mice after euthanasia and cut into small pieces. The testis samples were then cross-linked by 1% formaldehyde and terminated by glycine with a final concentration of 0.125 M. After homogenized, the samples were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.1% SDS) and the genomic DNAs were sonicated into fragments of 200–500 bp (Bioruptor Pico sonicator, high, 25cycle, 30s on/off). The DNA samples of 25 µg were incubated with protein A/G beads (36403ES25, Yeasen Biotechnology, Shanghai) conjugated with 2 µg primary antibody or IgG at 4 °C for overnight. After washed, the beads were used to extract genomic DNA with phenol-chloroform extraction methods. The primer sequences were described in Additional file 1: Table S2.

In total, 2.5 μg rabbit anti-Gm364 antibody, rabbit anti-Notch2 antibody, rabbit anti-NICD2 antibody, rabbit anti-MIB2 antibody, rabbit anti-DLL3 antibody, rabbit anti-AKT antibody, or rabbit anti-RICTOR antibody was first coupled to 30 μl protein A/G beads (Yeasen, Shanghai, China) for 4 h at 4 °C on a rotating wheel in 250 μl immunoprecipitate (IP) buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.05% Triton X-100, 0.05% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) with 1:100 protease inhibitor (Sigma) and 1:500 phosphatase inhibitor (Sigma). Meanwhile, 400 oocytes were lysed and ultrasonicated in 250 µl IP buffer and then pre-cleaned with 30 µl protein-A/G beads for 4 h at 4 °C. After that, protein A/G-coupled Rabbit IgG or specific antibodies were incubated overnight at 4 °C with pre-cleaned oocyte lysate supernatant. Finally, after being washed three times (10 min each with 250 µl IP buffer), the resulting beads with bound immunocomplexes were subjected to SDS-PAGE and western blot.