For TIRF microscopy, cortical cells were first transfected with tagBFP2-CAAX (plasmid #116856; Addgene, Watertown, MA, USA) [89 (link)] and pMH4-SYN-EGFP-ER (plasmid #22285; Addgene) [90 (link)]. The plasmid tagBFP2-CAAX encodes a fusion protein comprising blue fluorescence protein (BFP) and the CAAX motif-containing amino acids 172–184 of the proto-oncogene H-Ras, and the plasmid pMH4-SYN-EGFP-ER encodes a fusion protein comprising enhanced green fluorescence protein (EGFP), the ER-targeting sequence of calreticulin, and the ER retention sequence KDEL.
Then, cells were fixed in 4% formaldehyde and subjected to proximity ligation assay. Finally, cells were kept in HEPES buffer, and images were taken with a Nikon Eclipse Ti spinning disk microscope equipped with a Nikon CFI Apo TIRF 100× 1.49 N.A. oil objective using 405 nm, 488 nm, and 561 nm lasers. Images were recorded using Visitron Systems software (Visitron Systems, Puchheim, Germany), and numbers of red dots were quantified using ImageJ.
Then, cells were fixed in 4% formaldehyde and subjected to proximity ligation assay. Finally, cells were kept in HEPES buffer, and images were taken with a Nikon Eclipse Ti spinning disk microscope equipped with a Nikon CFI Apo TIRF 100× 1.49 N.A. oil objective using 405 nm, 488 nm, and 561 nm lasers. Images were recorded using Visitron Systems software (Visitron Systems, Puchheim, Germany), and numbers of red dots were quantified using ImageJ.
Free full text: Click here
