Rabbit igg isotype control

The ChIP assay was performed according to company protocol (Upstate) with some modification. Cells were first fixed in 1% formaldehyde and then quenched by 125 mM glycine. Cells were collected and resuspended in 1% SDS lysis buffer, and then sonicated to shear DNA into 100–1000 bp fragments. The supernatant was collected and subjected to overnight immunoprecipitation with 4 µg H3K27ac antibody (Abcam, Cambridge, UK), 25 µg HDAC3 antibody (Abcam, Cambridge, UK), or rabbit IgG isotype control (Novus Biological, Littleton, CO, USA) as a mock control, together with Magna ChIP™ Protein A + G Magnetic Beads (Millipore, Burlington, MA, USA) at 4 °C in a rotatory shaker, followed by reverse cross-link and protease K digestion. Eluted DNA was then purified using a MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and analysed by real-time PCR. Primer sequences are listed in Table 2.

The antibodies against ESR1 were rabbit polyclonal antibody HC-20 (against the C-terminus of the human protein) (sc-543, Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and mouse monoclonal antibody MA1-310 (against the synthetic peptide within the DNA-binding domain of the human protein) (Thermo Fisher Scientific, IL, USA). The antibodies against ESR2 were rabbit polyclonal antibody H-150 (against amino acids 1–150 of the human protein) (sc-8974, Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and mouse monoclonal antibody 6A12 MA1-23221 (against amino acids 1–153 of the human protein) (Thermo Fisher Scientific, IL, USA). The antibodies against GPER1 were rabbit polyclonal antibody K-19 (against the internal region of human GPR30) (sc-48524-R, Santa Cruz Biotechnology, Inc. Heidelberg, Germany) and rabbit polyclonal antibody H-300 (against amino acids 76–375 of human GPR30) (sc-134576, Santa Cruz Biotechnology, Inc. Heidelberg, Germany). The controls used in the experiments were rabbit IgG isotype control (Novus Biological, Centennial, CO, USA) and mouse IgG1/IgG2 isotype control (EXBIO, Vestec, Czech Republic).

MII oocytes were microinjected with 10 μM purified recombinant proteins (wild type α-SNAP, mutant α-SNAP L294A, wild type NSF and mutant NSF D1EQ) diluted in PBS if necessary. Anti-α-/β-SNAP, anti-γ-SNAP, and anti-NSF antibodies (Synaptic Systems) were microinjected at the maximum possible concentration. Anti-α-/β-SNAP, anti-γ-SNAP, mouse IgG isotype control (Novus Biologicals), and rabbit IgG isotype control (Novus Biologicals) were microinjected at 1 μg/μl. All antibodies and isotype controls were prepared in PBS. Microinjection pipettes were made by pulling borosilicate-glass capillary tubing in a mechanical puller (model P-97; Sutter Instrument Co., Novato, CA). Microinjections were performed using micromanipulators (Narishige) coupled to a Olympus IX-51 inverted microscope (Olympus). All micromanipulations were carried out in 5 μl drops of MEM/HEPES. For microinjection, needles were filled with injection solutions at the indicated concentrations, and about 7–10 pl was injected into the cytoplasm of MII oocytes by pneumatic pressure using a Pico-Injector (model PLI-100, Harvard Apparatus, Holliston, MA). Injected oocytes were used in CG exocytosis experiments after at least 1 h incubation in M16 medium, in a humidified atmosphere with 5% CO₂ at 37°C. The number of oocytes used for each experiment is indicated in the figure legends.