ATL2-1 is the major atlastin in HEK293T cells. (A) Total RNA was extracted from HEK293T cells, and cDNA was generated by RT-PCR. Real-time quantitative RT-PCR was performed to quantify the transcripts of human atlastins. All reactions were conducted in duplicate, and amplification signals from target genes were normalized to that from β-actin. The data represent the means ± SEM (error bars; n = 3). (B) The efficiency of in vitro fusion of ER microsomes isolated from HEK293T cells. Gluc1 and Gluc2 microsomes were mixed and incubated in the presence of GTP with increasing concentrations of PEG8000 at 37°C for 90 min. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments. (C) An anti-ATL2 antibody inhibits fusion by specifically recognizing ATL2. Donor and acceptor ATL2-bearing liposomes were incubated with an affinity-purified anti-ATL2 antibody (0.25 μM) at 30°C for 10 min. Fusion reactions were initiated by adding GTP and further incubated in the absence or presence of the N-terminal cytosolic domain of ATL2 at a subinhibitory concentration (1 µM) for 30 min. Fusion is expressed as the percentage of total fluorescence. The data represent the means ± SEM (error bars; n = 3). (D) Donor and acceptor ATL2- or ATL3-bearing liposomes were incubated with the indicated concentrations of an affinity-purified anti-ATL2 antibody in the presence of GTP at 30°C for 40 min. The data represent the means ± SEM (error bars; n = 3). n refers to the number of independent experiments.