Model ix71

Cells were adhered to glass coverslips, treated with PEME buffer for 5 min at room temperature, and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were washed once with PBS, incubated with the blocking buffer (3% BSA in PBS) for 1 h at room temperature, and then incubated with the primary antibody for 1 h at room temperature. The following primary antibodies were used: anti-CIF1 polyclonal antibody (1:1,000 dilution) (Zhou et al., 2018a (link)), anti-CIF2 polyclonal antibody (1: 1,000 dilution) (Zhang et al., 2019 (link)), anti-Protein A (anti-ProtA) polyclonal antibody (1:400 dilution) (Sigma-Aldrich), and FITC-conjugated anti-HA antibody (1:400 dilution) (Sigma-Aldrich). Cells were washed three times with PBS containing 0.1% Triton X-100. Except for the FITC-conjugated anti-HA antibody, cells were subsequently incubated with Cy3-conjugated anti-rabbit IgG at room temperature for 1 h. The slides were mounted in VectaShield mounting medium (Vector Labs) containing DAPI and examined using an inverted microscope (Model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60 × 1.42-NA DIC objective. Images were acquired and processed using the Slidebook5 software (Intelligent Imaging Innovations, Inc).

Experiments were performed on human embryonic kidney 293T cells (hereafter HEK-293T cells; American Type Culture Collection, Rockville, MD, USA) grown in Dulbecco modified Eagle's medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 μg/ml streptomycin in a humidified 5% CO₂ atmosphere at 37 °C. These cells express endogenously several subtypes of metabotropic P2Y receptors [29 (link),8 (link),6 (link)], but not P2X4R [38 (link)]. Cells were cultured in 75 cm² plastic culture flasks (NUNC, Rochester, NY) for 36–72 h, until they reached 80–95% confluence. Before the day of transfection, ~150,000 cells were plated on 35 mm culture dishes (Sarstedt, Newton, NC) and incubated at 37 °C for at least 24 h. For each culture dish of HEK-293T cells, transfection of either wild type or mutant receptors was conducted using 2 μg of DNA with 2 μl of jetPRIME™ reagent in 2 ml of Dulbecco modified Eagle's medium, according to the manufacturer's instructions (PolyPlus-transfection, Illkirch, France). After 24–48 h of incubation, the transfected cells were mechanically dispersed and re-cultured on 35 mm dishes of Corning 3294 CellBIND Surface for 2 – 8 hours before recording. Transfected cells were identified by the fluorescence signal of GFP using an inverted research microscope with fluorescence illuminators (Model IX71; Olympus, Melville, NY).