Journal: Science advances

Article Title: MAVI1, an endoplasmic reticulum-localized microprotein, suppresses antiviral innate immune response by targeting MAVS on mitochondrion.

doi: 10.1126/sciadv.adg7053

Figure Lengend Snippet: Fig. 4. MAVI1 is an ER membrane protein. (A and B) The SP (1 to 24 amino acids) (A) and TM domain (30 to 52 aa) (B) in MAVI1 predicted by SignaIP-4.1and TMHMM website, respectively, are shown. (C) HEK293 cells transfected with GFP-tagged MAVI1 were stained with Calnexin (ER maker), COXIV (mitochondria maker), EEA1 (en- dosome maker), LAMP2 (lysosome maker), RPL4 (ribosome maker), Vimentin (cytoskeleton maker), or Golgin-97 (Golgi apparatus marker) (Red). Nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI) (blue). (D) Schematic representation of Flag-tagged, full length (FL) MAVI1, SP-deleted (△SP) MAVI1, and MAVI1 mutant with the SP cleavage site mutated (A > W) is shown. (E) HEK293 cells transfected with empty vector (CTL) or expression vectors as described in (D) were subjected to IB analysis. (F) HEK293 cells stably expressing Flag-tagged MAVI1 were subjected to cellular fraction, followed by IB analysis. W.C.L., whole cell lysate; Cyto., cytoplasmic fraction; Mem., membrane fraction. (G) HEK293 cells stably expressing Flag-tagged MAVI1 were subjected to membrane interaction assay, and the supernatant was treated with or without 0.1 M Na2CO3, 0.1 M NaOH, or 1 M NaCl. The resultant soluble solution and pellet were subjected to IB analysis. (H) HEK293 cells stably expressing Flag- tagged MAVI1 were subjected to cellular fractionation, and membrane fractions were treated with or without proteinase K in the presence or absence of Triton X- 100, followed by IB analysis. (I) Membrane topology of calnexin, GM130, and MAVI1 proteins is shown.

Article Snippet: Cells were washed with phosphate-buffered saline (PBS) buffer for three times and then fixed with 4% paraformaldehyde in PBS buffer for 15 min and then permeabilized with 0.1% Triton X-100 in PBS buffer on ice for 10 min. After rinsing with PBS buffer for three times, blocking solution (5% bovine serum albumin in PBS buffer) was applied for 30 min and the corresponding primary antibody (anti-Calnexin (Proteintech, 10427-2-AP; 1:200 dilution), anti-COXIV (Proteintech, 11242-1-AP; 1:100), anti- EEA1 (home-made; 1:200 dilution), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822; 1:200 dilution), anti-RPL4 (Santa Cruz Biotechnology, sc-100838; 1:200 dilution), anti-Vimentin (Santa Cruz Biotechnology, sc-373717; 1:200 dilution), or anti-Golgi 97 (Santa Cruz Biotechnology, sc-59820; 1:200 dilution) was added in blocking buffer at 4°C overnight.

Techniques: Membrane, Transfection, Staining, Marker, Mutagenesis, Plasmid Preparation, Expressing, Stable Transfection, Cell Fractionation

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Figure Lengend Snippet: Five-day-old islets were infected with GBS and stained for endosomal markers. (A) Co-localization of invading streptococci (Blue) with the early endosomal marker EEA-1 (Green) and actin filaments (Red) just below the cell membrane at 30 min after infection. Scale bar = 10μm. EEA-1 was stained using rabbit polyclonal antibodies followed by Alexa Fluor 488-conjugated anti-rabbit IgG. The right panels are magnifications of the area indicated by the dashed rectangle. Scale bar = 1μm. (B) Colocalization of GBS (Blue) with acidic endosomes (Red) at 2 h post-infection. White arrows indicate bacteria. The right panels are magnifications of the area indicated by the dashed rectangle. Scale bar = 1μm. (C) CFU counts in cell lysates at different times after treatment of 5-day-old Caco-2 islets with antibiotics to kill extracellular bacteria. Shown are means ± SD of three independent experiments conducted in triplicate. *, p<0.05; by one-way Anova and Bonferroni test.

Article Snippet: Rabbit polyclonal anti-EEA-1 antibodies (ab2900) and goat polyclonal anti-rabbit IgG Alexa Fluor 488-conjugated secondary antibodies (ab15007) were obtained from Abcam, Cambridge, UK.

Techniques: Infection, Staining, Marker