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. 2020 Dec 29;133(24):jcs245571. doi: 10.1242/jcs.245571

Fig. 2.

Fig. 2. Sec71 is the only BFA target in BFA body formation in S2 cells. (A) Immunostaining of cells expressing V5::Sec71 (upper panels) or V5::Sec71E677K (lower panels) by anti-V5 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies. (B) Immunostaining of cells expressing V5::Garz (upper panels) or V5::GarzE740K (lower panels) by anti-V5 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies. (C) Immunostaining of cells transfected with double-stranded RNA against Sec71 (panels in middle row) or Garz (lower panels) or not transfected (upper panels) by anti-p120 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies. (D–I) Left, immunostaining of cells expressing V5::Sec71 (D), V5::Garz (E), V5::Sec71E677K (F), mTq2::Rab11 (green) (I), or transfected with double-stranded RNA against Sec71 (G–I). Anti-V5 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies (D–F). Anti-p120 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies (G). Anti-p120 (green), anti-GM130 (red), and anti-Golgin245 (blue) antibodies (H). Anti-GM130 (red) and anti-Rab6 (blue) antibodies (I). Right, plots of signal intensities from images on the left. Signal intensities were measured along the 1.5 µm (D,E) and 5 µm (F–I) arrows shown in insets. Graphs show the overlap between channels. (J,K) Electron micrographs of cells co-expressing GalT::APEX2::EGFP and V5::Sec71E677K (J) or V5::GarzE740K (K). GalT::APEX2::EGFP was visualized by osmium-enhanced DAB-deposition. (L) Percentages of wild-type, Sec71M717L and Sec71F713Y S2 cells with BFA bodies after 2 h of incubation with 0, 5, 10, 20, 30 and 50 μM BFA. BFA bodies were defined as focused Rab6 staining surrounded by GM130 staining. Results are mean±s.d. for n>250 in three independent experiments. (M) Immunostaining of wild-type cells (left) and genome-edited cells expressing BFA-resistant mutant Sec71M717L (right) treated with 50 μM BFA by anti-GM130 (green) and anti-Rab6 (red) antibodies. (N) Immunostaining of wild-type cells (left) and genome-edited cells with BFA-hypersensitive mutant Sec71F713Y (right) treated with 5 μM BFA with anti-GM130 (green) and anti-Rab6 (red) antibodies. Scale bars: 5 μm (A–C), 1 μm (D–I), 2 μm (J,K, left), 500 nm (J,K, right), 20 μm (M,N). **P<0.01, ***P<0.001 (unpaired two-tailed Student's t-test).

Sec71 is the only BFA target in BFA body formation in S2 cells. (A) Immunostaining of cells expressing V5::Sec71 (upper panels) or V5::Sec71E677K (lower panels) by anti-V5 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies. (B) Immunostaining of cells expressing V5::Garz (upper panels) or V5::GarzE740K (lower panels) by anti-V5 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies. (C) Immunostaining of cells transfected with double-stranded RNA against Sec71 (panels in middle row) or Garz (lower panels) or not transfected (upper panels) by anti-p120 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies. (D–I) Left, immunostaining of cells expressing V5::Sec71 (D), V5::Garz (E), V5::Sec71E677K (F), mTq2::Rab11 (green) (I), or transfected with double-stranded RNA against Sec71 (G–I). Anti-V5 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies (D–F). Anti-p120 (green), anti-GM130 (red), and anti-Rab6 (blue) antibodies (G). Anti-p120 (green), anti-GM130 (red), and anti-Golgin245 (blue) antibodies (H). Anti-GM130 (red) and anti-Rab6 (blue) antibodies (I). Right, plots of signal intensities from images on the left. Signal intensities were measured along the 1.5 µm (D,E) and 5 µm (F–I) arrows shown in insets. Graphs show the overlap between channels. (J,K) Electron micrographs of cells co-expressing GalT::APEX2::EGFP and V5::Sec71E677K (J) or V5::GarzE740K (K). GalT::APEX2::EGFP was visualized by osmium-enhanced DAB-deposition. (L) Percentages of wild-type, Sec71M717L and Sec71F713Y S2 cells with BFA bodies after 2 h of incubation with 0, 5, 10, 20, 30 and 50 μM BFA. BFA bodies were defined as focused Rab6 staining surrounded by GM130 staining. Results are mean±s.d. for n>250 in three independent experiments. (M) Immunostaining of wild-type cells (left) and genome-edited cells expressing BFA-resistant mutant Sec71M717L (right) treated with 50 μM BFA by anti-GM130 (green) and anti-Rab6 (red) antibodies. (N) Immunostaining of wild-type cells (left) and genome-edited cells with BFA-hypersensitive mutant Sec71F713Y (right) treated with 5 μM BFA with anti-GM130 (green) and anti-Rab6 (red) antibodies. Scale bars: 5 μm (A–C), 1 μm (D–I), 2 μm (J,K, left), 500 nm (J,K, right), 20 μm (M,N). **P<0.01, ***P<0.001 (unpaired two-tailed Student's t-test).