Journal:

Article Title: Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

doi: 10.1038/emboj.2008.177

Figure Lengend Snippet: Mutations impairing Ub binding alter the localization of Rabex-5. HeLa cells were transiently transfected and fixed with 4% formaldehyde ∼24 h after transfection. Fixed cells were incubated with mouse anti-myc and Alexa 568-conjugated anti-mouse antisera. The substitutions in the ZnF and MIU domains interfere with Ub binding to Rabex-5 (Lee et al, 2006; Mattera et al, 2006; Penengo et al, 2006). myc–Rabex-5 wild type (WT) (A) is predominantly recruited to large (∼2 μm) vesicles that are positive for the early endosomal marker EEA1 (see Figure 3); in contrast, expression of comparable levels of myc–Rabex-5 Ub-binding mutants, such as Δ13–49 (deletion of the ZnF) (B), A58D (C) and Y25A/A58D (D), results in predominantly cytosolic accumulation (some threads and membrane ruffles are also visible). Enlarged vesicles containing the recombinant proteins were observed in 99, 2, 1 and 1% of cells expressing intermediate to high levels of myc–Rabex-5 WT or the Δ13–49, A58D and Y25A/A58D mutants, respectively (n=396, 643, 534 and 372, respectively). Approximately 8–12% of cells transfected with either Rabex-5 A58D or Rabex-5 Y25A/A58D also exhibited large artificial aggregates containing the recombinant proteins that were clearly discernable from the vesicles containing myc–Rabex-5 WT; this aggregation was less frequent (0–3%) in cells transfected with the ΔZnF or Y25A/Y26A mutants. Scale bars=10 μm.

Article Snippet: For immunofluorescence microscopy, we used mouse anti- myc (9E10; Covance Research Products, Berkeley, CA), rabbit anti- myc (gift from RS Hegde, CBMP, NICHD), chicken anti- myc (Molecular Probes, Eugene, OR), mouse anti-EEA1 (BD Biosciences, San Jose, CA), mouse anti-HA (HA.11; Covance Research Products), rabbit anti-HA (Affinity BioReagents, Golden, CO) and rabbit anti-GFP (Molecular Probes).

Techniques: Binding Assay, Transfection, Incubation, Marker, Expressing, Recombinant

Journal:

Article Title: Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

doi: 10.1038/emboj.2008.177

Figure Lengend Snippet: Recruitment of myc–Rabex-5 WT but not Ub-binding mutants to early endosomes. HeLa cells were transfected and fixed as described in the legend to Figure 2. Fixed cells were incubated with rabbit anti-myc and mouse anti-EEA1 antisera, followed by incubation with Alexa 568-conjugated anti-rabbit and Alexa 488-conjugated anti-mouse antisera. Rabex-5 WT (A–C) is recruited to early endosomes, whereas the Rabex-5 Ub-binding mutants (D–R) exhibit a predominantly cytosolic distribution. Although expression of all constructs results in enlargement of early endosomes as compared with untransfected cells, only myc–Rabex-5 WT is recruited to these structures. The enlargement of early endosomes in cells transfected with myc–Rabex-5 WT was greater than in cells expressing the Ub-binding mutants. In some instances, particularly for Rabex-5 Y25A/Y26A (G–I, insets), it was possible to observe decoration of enlarged early endosomes by the Ub-binding mutants amidst their predominantly cytosolic distribution. Insets in (G–I) represent a × 3 magnification of areas in the dotted squares. (C, F, I, L, O, R) Merged images of the panels at their left. Scale bars=10 μm.

Article Snippet: For immunofluorescence microscopy, we used mouse anti- myc (9E10; Covance Research Products, Berkeley, CA), rabbit anti- myc (gift from RS Hegde, CBMP, NICHD), chicken anti- myc (Molecular Probes, Eugene, OR), mouse anti-EEA1 (BD Biosciences, San Jose, CA), mouse anti-HA (HA.11; Covance Research Products), rabbit anti-HA (Affinity BioReagents, Golden, CO) and rabbit anti-GFP (Molecular Probes).

Techniques: Binding Assay, Transfection, Incubation, Expressing, Construct

Journal:

Article Title: Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

doi: 10.1038/emboj.2008.177

Figure Lengend Snippet: Colocalization of myc–Rabex-5 and (HA)3–Ub on early endosomes; interference of Rabex-5 recruitment to early endosomes by (HA)3–UbΔG75/76. Cells were co-transfected with myc–Rabex-5 and (HA)3–Ub or (HA)3–UbΔG75/76 and fixed 24 h after transfection. Fixed cells were incubated with chicken anti-myc, mouse anti-EEA1 and rabbit anti-HA, followed by incubation with Alexa 594-conjugated anti-chicken, Alexa 488-conjugated anti-mouse and Alexa 647-conjugated anti-rabbit antisera. Arrows in A–D show colocalization of myc–Rabex-5 with (HA)3–Ub WT on early endosomes. We observed this phenotype in 90 and 28% of cells co-transfected with (HA)3–Ub or (HA)3–UbΔG75/76, respectively (n=104 and 421 cells, respectively, exhibiting medium–high expression levels of myc–Rabex-5). The phenotype in >70% of cells co-transfected with (HA)3–UbΔG75/76 is shown in (E–H). (D, H) Merged images of the preceding panels. Scale bars=10 μm.

Article Snippet: For immunofluorescence microscopy, we used mouse anti- myc (9E10; Covance Research Products, Berkeley, CA), rabbit anti- myc (gift from RS Hegde, CBMP, NICHD), chicken anti- myc (Molecular Probes, Eugene, OR), mouse anti-EEA1 (BD Biosciences, San Jose, CA), mouse anti-HA (HA.11; Covance Research Products), rabbit anti-HA (Affinity BioReagents, Golden, CO) and rabbit anti-GFP (Molecular Probes).

Techniques: Transfection, Incubation, Expressing

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