Journal: The Journal of Biological Chemistry

Article Title: Myristoylated methionine sulfoxide reductase A is a late endosomal protein

doi: 10.1074/jbc.RA117.000473

Figure Lengend Snippet: Myristoylated MSRA is anchored to the membrane of late endosomes. A and C, COS7 cells were transiently transfected with three constructs, Stard3-FLAG, mRFP-Rab7, a late endosomal protein, and either (A) myristoylated MsrA-gfp or (C) nonmyristoylated MsrA-gfp. After 8 h, cells were treated with 20 mm NH4Cl for 16 h to alkalinize and enlarge late endosomes and lysosomes. The images within the white box of the upper panels are magnified in the lower panels. Scale bar, 10 μm. B and D, the merged images were scanned along the white arrow for relative fluorescence intensity with ZEN 2 software. The black arrows indicate the direction of scanning.

Article Snippet: The Met-307 and M307R/N311D mutants of the START domain were generated with the QuikChange kit. pDsRed2-Mito was purchased from Clontech (number 6975-1). mRFP-RAB7 was a gift from Ari Helenius (Addgene plasmid number 14436).

Techniques: Transfection, Construct, Fluorescence, Software

Journal: The Journal of Biological Chemistry

Article Title: Myristoylated methionine sulfoxide reductase A is a late endosomal protein

doi: 10.1074/jbc.RA117.000473

Figure Lengend Snippet: Subcellular fractionation demonstrates that myristoylated MSRA is enriched in the late endosome fraction. A, myristoylated MSRA is most prominent in fraction 1 from wildtype (WT) mouse liver and is not present in fractions prepared from the MSRA knockout liver (KO). Fraction 1 also contains most of the intact late endosomes. Each fraction was evaluated by immunoblot with these marker proteins: RAB7 (late endosome), mtHSP70 (mitochondria), myristoylated MSRA, total MSRA, and STARD3. The asterisk (*) indicates a nonspecific band. The molecular size markers of protein are indicated in kDa. The specificity of the mAb for the myristoylated form of MSRA is shown in panel B. 100 ng of purified, recombinant human myristoylated or nonmyristoylated MSRA were subjected to SDS-PAGE followed by immunoblotting by either the anti-myristoylated MSRA antibody or the general anti-MSRA antibody. As indicated under “Experimental procedures,” the concentration of protein was determined spectrophotometrically. However, to assure that equal amounts of the two proteins were loaded for immunoblotting, we also subjected 1 μg of each protein SDS-PAGE followed by staining with Coomassie Brilliant Blue. C, the anti-myristoylated MSRA and general anti-MSRA antibodies are specific for MSRA. This was demonstrated by immunoblotting homogenates of liver and kidney from WT and MSRA knockout mice. The asterisk (*) marks nonspecific bands detected by the general anti-MSRA antibody. Tubulin served as a loading control.

Article Snippet: The Met-307 and M307R/N311D mutants of the START domain were generated with the QuikChange kit. pDsRed2-Mito was purchased from Clontech (number 6975-1). mRFP-RAB7 was a gift from Ari Helenius (Addgene plasmid number 14436).

Techniques: Fractionation, Knock-Out, Western Blot, Marker, Purification, Recombinant, SDS Page, Concentration Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 1. Intracellular distribution of Rab7 in osteoclasts. A, cells were cul- tured on glass coverslips for 16 h and then were incubated with anti-Rab7 antibody. Images show one optical section of Rab7 labeling in a non-resorbing osteoclast (a) and in mononuclear cells (b). B, double staining of Rab7 and Lamp2 in mouse osteoclasts. C, osteoclasts were cultured on bovine bone slices for 48 h. Double labeling of Rab7 with F-actin (a–c) and 3-integrin (d–f) demonstrates that Rab7 is predominantly localized at the ruffled border membrane in resorbing oste- oclasts. Boxed region in (f) is enlarged (g) to demonstrate the localization of Rab7 and 3-integrin at the ruffled border. Lo- calization of Rab7 and v-H-ATPase in resorbing osteoclasts is shown in D. Bars: A, B, and D, 10 m; C, c and f, 10 m; C, g, 2 m.

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Incubation, Labeling, Double Staining, Cell Culture, Membrane

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 2. The Rab7 antisense ODNs reduces Rab7 protein and mRNA expression. A and B are Western blots of Rab7 and rab11 proteins after a 48-h incubation with 5 M Rab7 sense and antisense ODNs. Cell lysates from 6 slices of each group were separated by 15% SDS- polyacrylamide gel electrophoresis and immunoblotted with anti-Rab7 (A) and anti-rab11 (B) antibodies and developed using ECL. The blots were stripped and reprobed with a monoclonal anti-actin antibody and developed in the same way. C is the analysis of Rab7 mRNA by reverse transcription-PCR after a 48-h incubation with 5 M Rab7 sense and antisense ODNs. The gels were quantitated by densitometric analysis, normalized versus actin, and presented in arbitrary units as percentage with respect to control. C, control; S, sense ODNs; A, antisense ODNs. The intensity of immunofluorescent staining for Rab7 (D) in antisense (Ant)-treated osteoclasts was clearly weaker than in controls (Con) or sense (Sen)-treated cells. Transmission images in D show resorption pits beneath osteoclasts. Original magnification, 400.

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Western Blot, Incubation, Polyacrylamide Gel Electrophoresis, Reverse Transcription, Control, Staining, Transmission Assay

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 3. Effects of Rab7 ODNs on osteoclastic bone resorption. A, cells were cultured on bovine bone slices and incubated with Rab7 sense and antisense ODNs (0.1–10 M) for 48 h. The cells were fixed with 3% paraformaldehyde, and the nuclei were stained with Hoechst 33258. Multinucleated cells with more than three nuclei were counted and presented as a percentage rate versus untreated cultures. B, microscopic images of resorption pits stained with horseradish peroxidase-conjugated wheat germ agglutinin in untreated and 5 M Rab7 sense- or antisense ODN-treated cultures. Con, control; Sen, sense ODNs; Ant, antisense ODNs. Original magnification, 100. Total resorbed area (C), the number of pits (D), and average pit area (E) on each bone slice were measured and expressed as a percentage from untreated cultures. Carboxyl-terminal telopeptide of type I collagen (CTx) concentration (F) in culture medium was significantly lower in antisense ODN-treated cultures. Data are shown as the mean S.D. of six bone slices. ***, p 0.001. Black bars, no ODN controls; gray bars, sense ODNs; white bars, antisense ODNs.

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Cell Culture, Incubation, Staining, Control, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 4. Effects of Rab7 ODNs on os- teoclast polarization and actin orga- nization. A, cells were cultured on bovine bone slices and incubated with Rab7 sense and antisense ODNs (0.1–10 M) for 48 h. The cells were fixed with 3% paraformaldehyde. The nuclei were stained with Hoechst 33258, and the F- actin was stained with fluorescein isothio- cyanate-labeled phalloidin. The number of multinucleated cells (MNC) containing the actin ring was counted and expressed as a percentage from untreated cultures. Data are shown as the mean S.D. of six bone slices. **, p 0.01; ***, p 0.001. Black bars, no ODN controls; gray bars, sense ODNs; white bars, antisense ODNs. B, F-actin organization in an osteoclast from control cultures (a–c) and from Rab7 antisense ODN-treated cultures. a and c are single optical section at the bone sur- face level, d is at the nuclei level, b and f are vertical confocal images. The solid lines in b and f demarcate the bone sur- face. The white arrowheads in b show the accumulation of F-actin at the sealing zone. c and g show transmission images of the resorption pits beneath the oste- oclasts. The black arrowheads in c dem- onstrate the edge of resorption pit. Bars, 10 m.

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Cell Culture, Incubation, Staining, Labeling, Control, Transmission Assay

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 5. Disrupted localization of v-HM-ATPase at the ruffled border in Rab7 antisense ODN-treated oste- oclasts. A, confocal microscopic images at the bone surface level show that v-H- ATPase is localized at the ruffled border of a resorbing osteoclast in control or sense ODN-treated cultures. B, confocal microscopic images at the nuclei level (a–c) and the bone surface level (d–f) dem- onstrate that in an antisense ODN- treated osteoclast, v-H-ATPase is dis- tributed in the cytoplasm and the basal plasma membrane. Bars, 10 m.

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Control, Clinical Proteomics, Membrane

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 6. The inhibition of transferrin transportation to the ruffled border in Rab7 antisense ODN-treated oste- oclasts. A, in control cultures, trans- ferrin was endocytosed and transported quickly to the ruffled border membrane inside the actin ring of resorbing oste- oclasts. B, in Rab7 antisense ODN- treated osteoclasts, the uptake of trans- ferrin and its transportation into the intracellular vesicles were not disturbed, but its localization at the ruffled border membrane disappeared. Images are sin- gle optical section at the nuclear level (a) and bone surface level (b). Bars, 10 m. C shows the quantification analysis of transferrin transportation to the ruffled border. The number of osteoclasts (OC) with the appearance of transferrin (Tf) at the ruffled border (RB) was counted and expressed as a percentage from untreated cultures. ***, p 0.001

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Inhibition, Control, Membrane

Journal: Journal of Biological Chemistry

Article Title: Downregulation of Small GTPase Rab7 Impairs Osteoclast Polarization and Bone Resorption

doi: 10.1074/jbc.m010999200

Figure Lengend Snippet: FIG. 7. Ultrastructural changes of the ruffled border mem- brane induced by down-regulation of Rab7. Transmission electron microscopic images of the osteoclast in control culture (a) and antisense ODN-treated culture (b). Each image was chosen from five consecutive sections of the same cell. n, nucleus; RB, ruffled border; SZ, sealing zone. Original magnification: 3000 (a), 4000 (b).

Article Snippet: Goat polyclonal anti-Rab7 antibody was purchased from Santa Cruz Biotechnology, Inc.

Techniques: Transmission Assay, Control