Pa5 47012

The EECs were fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 10 min. After two PBS washes, the cells were permeabilized with 0.1% Triton 100-X (Beyotime Institute of Biotechnology) at room temperature for 30 min. The EECs were then washed with PBS three times and blocked with blocking buffer (P0260, Beyotime Institute of Biotechnology) at 37°C for 30 min. Samples were then incubated with the primary antibodies diluted in blocking buffer overnight at 4°C, followed by incubation for secondary antibodies for 1 h. The primary antibodies used included anti-MAP1LC3B (ab51520, Abcam), anti-NPR3 (ab97389, Abcam), anti-CDH11 (H00001009, Novus), anti-PLXND1 (PA5-47012, Invitrogen), and anti-ORAI1 (ab244352, Abcam). The secondary antibodies used were FITC-labeled goat anti-rabbit IgG (H + L) (A0423, Beyotime Institute of Biotechnology) and Cy3-labeled goat anti-mouse IgG (H + L) (A0521, Beyotime Institute of Biotechnology). The cells were then digitalized on a Leica SP5 confocal microscope (Leica Microsystems, Germany) and analyzed using Image-Pro Plus 5.0 (Media Cybernetics).

For western blotting experiments, EEC lysates were prepared from in vivo samples, and immunoblot analyses were performed. EECs were scraped off the plates and lysed in RIPA buffer. Total proteins were detected using the BCA assay (P0012, Beyotime Biotechnology), resolved in running buffer by SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (Millipore) by wet blotting at 100 V for 1 h. Twenty microgram protein samples were mixed with 4X Laemmli buffer (Omp-02, Omiget) and denatured at 95°C for 10 min. Membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with primary antibodies. The antibodies used were microtubule-associated protein 1 light chain three beta (MAP1LC3B; ab192890), PLXND1 (PA5-47012, Invitrogen) and actin beta (ACTB; ab8226, Abcam). Membranes were then incubated with HRP-conjugated secondary antibodies (A0208, Beyotime Biotechnology), visualized by chemiluminescence detection, and quantified using Image QuantTL software (GE Healthcare, Sweden).