Confocal laser scanning microscope

Primary rat VSMCs and arterial sections were collected for immunofluorescence staining. Briefly, VSMCs were fixed in 4% paraformaldehyde for 30 min, and arterial sections were repaired with antigen retrieval sodium buffer. Cells or arterial sections were permeabilized with 0.4% Triton X-100 (catalog no.: ST795; Beyotime) and blocked with 10% donkey serum (catalog no.: SL038; Solarbio, China) at room temperature. Samples were incubated with primary antibodies against RGMa (1:50 dilution; catalog no.: sc-393046; Santa Cruz), CD68 (1:100 dilution; catalog no.: ab201340; abcam, UK), SM22α (1:200 dilution; catalog no.: ab14106; abcam), α-SMA (1:200 dilution; catalog no.: 19245; CST, UK), and Slug (1:50 dilution; catalog no.: sc-166476; Santa Cruz) at 4°C overnight, followed by secondary antibodies Alexa Fluor 488 (1:200 dilution; catalog no.: SA00013-5; Proteintech, China) and 555 (1:100 dilution; catalog no.: A0460; Beyotime) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (catalog no.: C1002; Beyotime) for 10 min and protected by antifade mounting medium. Triple immunofluorescence staining was performed according to the manufacturer's instructions (catalog no.: AFIHC024; China). Images were captured using a confocal laser scanning microscope (Andor, UK) and analyzed by ImageJ software.

BiFC assay was performed in Nicotiana benthamiana leaves. The ORFs of TapLDδ and TaGApC2-6D without termination codons were recombined with C-terminal part of YFP in the pSPYCE-35S/pUC-SPYCE vector and N-terminal part of YFP in the pSPYNE-35S/pUC-SPYNE vector, respectively. The pSPYCE-TaPLDδ and pSPYNE-TaGAPC2-6D were co-transformed into Nicotiana benthamiana leaves with corresponding empty vectors as negative controls. The Nicotiana benthamiana leaves’ protoplasts were observed for fluorescence at 48 h after transformation using a confocal laser scanning microscope (Andor, Belfast, UK).