Fig. 6: In vitro reconstitution of AGPAT2- and DRP1-mediated ER tubulation.

a The ER was purified from WT, DRP1-KO, and AGPAT2-KO MEFs and incubated with 1 µM recombinant DRP1 in the absence of GTP for 3 h. The morphology of the ER membrane was analyzed by transmission electron microscopy with negative staining. b The length of ER tubules was quantified. Mean ± SD (n = 15 tubules). c GFP does not tubulates the ER in vitro. The ER isolated from Drp1-KO MEFs was incubated with 1 µM GFP for 3 h. d DRP1 tubulates the ER membrane in a dose-dependent manner in vitro. The ER isolated from Drp1-KO MEFs was incubated with varying amounts of DRP1 in the absence of GTP for 3 h. e The length of ER tubules was quantified. Mean ± SD (n = 15 tubules). f ER membranes purified from DRP1-KO MEFs were incubated with 1 µM recombinant WT DRP1 or DRP1(K38A, G350D) in the absence of GTP for 3 h. g The length of ER tubules was quantified. Mean ± SD (n = 10 tubules). (h) Tubule widths of purified ER (WT) and DRP1-induced tubules from DRP1-KO ER (WT and mutant) were quantified. Mean ± SD (n = 15 tubules). i ER membranes purified from WT MEFs were incubated with the indicated antibodies for 3 h. j The length of ER tubules was quantified. Mean ± SD (n = 10 tubules). Statistical analysis was performed using one-way ANOVA with post-hoc Tukey (b, e, g, h, j). Source data are provided as a Source Data file.