Anti flag magnetic beads

The Hc-hrg-1 coding sequence with a HA tag and the Hc-vha-2 sequence with a FLAG tag were cloned into the Hind III-EcoR I sites of the pcDNA3.1(+) vector (Takara Biomedical Technology). These plasmids were co-transfected into the HEK293T cells. At 24 h post-transfection, the cells were lysed in radioimmunoprecipitation (RIPA) lysis buffer (Fude Biological technology) containing 1 × protease inhibitor cocktail (Bimake, Houston, USA) at 4°C for 30 min, with constant rocking. The cellular debris was removed by centrifugation at 12,000 × g for 10 min at 4°C. The supernatant was incubated with 20 μl anti-FLAG magnetic beads (Bimake) overnight at 4°C, under constant agitation. Immunoprecipitated proteins bound to beads were concentrated using a magnetic separator, washed three times in PBST (NaCl 136.89 mM; KCl 2.67 mM; Na2HPO4 8.1 mM; KH2PO4 1.76 mM; 0.5% Tween-20) and eluted in 50μl sodium dodecyl sulfate (SDS) buffer (50 μl) containing (1.5% Tris, 9.4% glycine and 0.5% SDS). The immunoprecipitates and WCLs were analysed by Western blot probed with specific antibodies against HA or FLAG tag.

All possible interactions between proteins in vitro were examined using pull-down assay^{45 (link)}. wspR was cloned into pET30a with a C-terminal His-tag, while rpfG was cloned into pET30a fused to a C-terminal FLAG-tag. Both WspR and RpfG were expressed in E. coli BL21 (DE3) and induced by 0.8 mM IPTG. 1 mL of bacteria lysate containing WspR-His or RpfG-FLAG was then incubated with 10 μL of anti-FLAG magnetic beads (Bimake, Shanghai, China). After overnight incubation at 4 °C, the beads were washed 3 times for 5 min each with 1 ml of 10 mM PBS buffer (pH, 7.5) containing 1% Triton X-100. Proteins bound to the beads were eluted with 45 μl elution buffer (0.2 M glycine HCl, pH 3.0), followed by the addition of 5 μl neutralization buffer (1 M Tris, pH 10). The eluted samples were boiled in 4×SDS loading buffer for 8 min. These samples were loaded into SDS-PAGE for Western blotting, and proteins were detected using anti-FLAG (No. M20008S, Abmart) and anti-His-tag (No. M30111L, Abmart) from Shanghai, China. Uncropped and unprocessed scans of gels & blots were provided in Supplementary Fig. 13.

For Co-immunoprecipitation (Co-IP) assay, cells were collected 24 h after transfection and lysed in lysis buffer [1.0% (v/v) NP-40, 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 0.15 M NaCl] supplemented with a protease inhibitor cocktail (Sigma), and a phosphatase inhibitor cocktail (Sigma). After centrifugation for 10 min at 14,000 × g, supernatants were collected and incubated with the indicated antibodies followed by the addition of protein A/G beads (Santa Cruz), with Anti-Flag magnetic beads (Bimake), anti-Myc magnetic beads (Bimake), or anti-HA magnetic beads (Bimake). After incubation overnight at 4°, beads were washed four times with lysis buffer. Immunoprecipitates were eluted by boiling with 2×SDS loading buffer containing 100 mM Tris-HCl pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue, and 1% (v/v) 2-mercaptoethanol.

HEK293T cells in four well of a six-well plate were co-transfected with wild-type BMP15 and wild-type/mutant His₁₀-GDF9 (8g wild-type BMP15+8g wild-type His₁₀-GDF9; 8g wild-type BMP15+8g c.1283G>C His₁₀-GDF9; 8g wild-type BMP15 + 4g c.961C>T His₁₀-GDF9+ 4g c.1283G>C His₁₀-GDF9). Cells were lysed 48 hours after transfection using IP lysis buffer (87787, Thermo, USA) containing a protease-inhibitor cocktail (78425, Thermo, USA) for 30 min at 4℃. The protein concentration of each group of lysates was normalized to 1.5mg/ml. A portion of the lysates was used as the input. The remaining lysates were incubated with anti-Flag magnetic beads (B26102, Bimake, China) on a rocking platform overnight at 4℃, followed by magnetic separation. The immunoprecipitated complexes were washed with the PBST buffer three times, boiled in reducing protein sample loading buffer (LT101S, EpiZyme, China) for 10 min, and subjected to PAGE electrophoresis and western blotting after finally magnetic separation. Primary antibodies used were mouse anti-His-tag (1:1000, HA601079, Huabio, China) for detecting His₁₀-GDF9, rabbit anti-BMP15 (1:2000, 18982-1-AP, Proteintech, China) for detecting BMP15 precursor. The experiment was repeated 3 times.

Samples were fully lysed in cell lysis buffer containing protease inhibitors (FUDE Biological Technology, China) and centrifuged at high speed at 4°C. Supernatants were incubated with protein A/G magnetic beads (Bimake, United States) or anti-Flag magnetic beads (Bimake, United States) for 6–12 h at 4°C. protein A/G magnetic beads and anti-Flag magnetic beads were pre-blocked with 0.5% bovine serum albumin/PBS for 2 h, and protein A/G magnetic beads were pre-coupled with specific antibodies using a rotary instrument for 6–12 h at 4°C. After washing with cell lysis buffer, immunoprecipitants and loading buffer were boiled for 10 min at 100°C. 10% of the supernatant was served as input. Immunoprecipitants were further analyzed by western blot.

Tobacco leaves co-infiltrated with 35S::RMF1/2-FLAG and 35S::ASK1-MYC or 35S::RMF1/2-GFP and 35S::DMC1-FLAG were ground to a fine powder in liquid nitrogen and homogenized in protein lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 0.5% NP-40, 1% cocktail proteinase inhibitor). The supernatants were incubated at 4 °C for 1 h, and cell debris was removed by centrifugation at 21,130 × g for 10 min. After preclearing with anti-FLAG magnetic beads (Bimake, B26102) or anti-GFP magnetic beads (KT Life Technology, KTSM1334), the lysates were incubated with corresponding beads at 4 °C overnight. The immunoprecipitation complexes were washed three times with 1 ml protein lysis buffer. Proteins retained on beads were separated by SDS-PAGE and detected using anti-FLAG (GNI, GNI4110-FG, 1:2000 dilution), -MYC (Sigma-Aldrich, 05-724-25UG, 1:2000 dilution), or -GFP (GNI, GNI4110-GP, 1:2000 dilution) antibodies.