Fig. 3.

Purification of M7Vs. (A) Gradient centrifugation of endogenous M7Vs. PNS from SV40MES-13 cells was first separated on a sucrose gradient and then Western blotted for TRPM7, plasma membrane (Na⁺/K⁺ ATPase), ER (PDI: protein disulfide isomerase), and recycling endosome (Rab11) markers. (B) Postnuclear supernatants of HEK293 cells stably expressing TRPM7-HA (with GCaMP6s in the S1/2 loop) were isolated using mouse anti-HA–conjugated magnetic nanoparticles. Vesicles were visualized by incubation with Ca²⁺, and nanoparticles were detected with Alexa Fluor 546-conjugated anti-mouse secondary antibodies. (C) Anti-HA Western blot: HA-peptide blocks nanoparticle binding to vesicles. (D) Western blot of isolated vesicles: enrichment of vesicles and depletion of ER (Calnexin; Canx or PDI) and mitochondria (cyt-C). B, Bound; NB. Not bound. See also Fig. S3A.