Journal: Aging cell

Article Title: Fast anterograde transport of Herpes Simplex Virus: Role for the amyloid precursor protein of Alzheimer’s disease

doi:

Figure Lengend Snippet: APP and TGN46 are associated with motile GFP-labelled HSV. (A) Diagram of HSV indicating the viral and host cell compartments recognized by the antibodies. (B) Cellular proteins co-sediment with GFP-HSV. Motile virus preparations were separated into particulate and soluble proteins by centrifugation and the resultant supernatants (sup) and pellets (pel) probed for proteins representative of each compartment by immunoblotting. Both viral and cellular proteins are detected only in the pellet: viral capsid (VP5), tegument (VP22), envelope (gD) and the VP16-GFP label (GFP), together with TGN46, a trans-Golgi marker, and APP detected with two different antibodies, one against the peptide in the cytoplasmic domain, C-APP, and the other against the extracellular domain, N-APP. Both anti-APP antibodies detected a single, ~120-kDa band in the pellet that could be superimposed in stripped and re-probed blots. Synaptophysin and kinesin are also detected in pellets of viral particles. JIP is not detected in either the supernatant or the pellet from the virus, but is present in the Vero cells (additional third lane from left). (C) Immunofluorescence shows that the cellular proteins, TGN46 and APP, are associated with GFP-labelled viral particles. Preparations of motile VP16-GFP-labelled HSV stained for viral compartments, capsid (VP5), envelope (gD), as well as cellular proteins (TGN46 and C-APP). Antibodies appear red, VP16-GFP-labelled HSV is green and overlap appears yellow. The GFP-label was associated with all three viral compartments, demonstrating that labelled particles represent intact virus. Appropriately, capsid (VP5) was within or beside the GFP, and envelope (gD) surrounded GFP label. GFP-labelled particles were also found associated with C-APP and TGN46. C-APP was apparently exposed on the particle surface, as it was detected without fixation or detergent. Both anti-TGN46 and anti-C-APP stain two different structures: small particles containing a single virus and large cisternae with multiple virions. Percentiles indicate the average proportion of each type of structure in motile viral preparations. The scale bar, bottom right, is the same for all images. (D) Detergent treatment which removes anterograde motility also separates APP from labelled viral particles. Motile preparations of GFP-labelled HSV were treated with Triton-X100 under conditions known to deplete anterograde and enhance retrograde motility (Bearer et al., 2000). Supernatants (sup) and pellets (pel) were separated by centrifugation and protein composition analysed by immunoblotting in parallel with untreated virus as shown in (B). Viral particles retained proteins representative of capsid and tegument (VP5, VP22) and the GFP label, whereas membrane components were solubilized (gD, TGN46 and APP). As in (B), APP was detected with two different antibodies, both of which detect a single band in the supernatant.

Article Snippet: Antibodies included mouse anti-VP5 (Virusys), mouse anti-VP22 (Invitrogen), mouse anti-gD (QED Biosciences), sheep anti-TGN46 (Serotec), mouse anti-kinesin (SUK4, Cytoskeleton), anti-squid kinesin (Chemicon), anti-synaptophysin (Sigma), mouse anti-GFP (Clontech), rabbit anti-C-APP (Zymed) and goat anti-N-APP (BioDesign).

Techniques: Centrifugation, Western Blot, Marker, Immunofluorescence, Staining

Journal: Journal of Nanobiotechnology

Article Title: Zinc oxide nanosphere for hydrogen sulfide scavenging and ferroptosis of colorectal cancer

doi: 10.1186/s12951-021-01069-y

Figure Lengend Snippet: Transcriptome analysis in VZnO-treated HCT116. A KEGG pathway analysis based on the RNA-seq results in HCT116. B Representative heatmap of gene expression levels. C Representative scatter plot of 140 significant genes (33 unregulated genes marked in red and 107 downregulated genes marked in green) for Treat vs. Con. D mRNA levels of GGCT, RRM1 and RRM2 by RNA-seq. E Representative Western Blot result of GGCT, RRM1 and RRM2. Membranes were re-probed for GAPDH expression to show that similar amounts of protein were loaded in each lane

Article Snippet: Primary antibodies were used at the following dilutions: rabbit-anti-GPX4 (Sigma-SAB4300725) 1:2000; rabbit-anti-CBS (Sigma-AV45746) 1:4000; rabbit-anti-COX2 (Sigma-SAB4200576) 1:5000; goat-anti-NOX1 (Sigma-SAB2501686) 1:5000; rabbit-anti-TRF1 (Sigma-SAB4502943) 1:500; rabbit-anti-GAPDH (Invitrogen-PA1-987) 1:3000; rabbit-anti-GGCT (Invitrogen-PA5-80,658) 1:1000; rabbit-anti-RRM1 (Invitrogen-PA5-75,989) 1:1000; rabbit-anti-RRM2 (Invitrogen-PA5-27,856) 1:1000.

Techniques: RNA Sequencing Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

doi: 10.1371/journal.pone.0102817

Figure Lengend Snippet: (A to C) TM4SF5-null Chang (normal hepatocyte) and TM4SF5-expressing Chang-TGFβ1 (Chang cells chronically treated with TGFβ1) cells were analyzed for CD9, CD63, CD151, and TM4SF5 expression by flow cytometry (A), by RT-PCR (B), and by Western blot (C). Data represent three independent experiments.

Article Snippet: Double-immunostaining of human liver tissues were obtained from each patient as reported previously after informed content , or murine liver tissues were incubated with primary antibodies specific for TM4SF5 (Clone # 27, anti-TM4SF5 mAb, see above), CD63, and CD151 (Cell Signaling Technol.) with or without DAPI staining.

Techniques: Expressing, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

doi: 10.1371/journal.pone.0102817

Figure Lengend Snippet: (A to D) Liver tissues from mice administrated with control vehicle or CCl 4 every other day for 2 weeks were used for RT-PCR analysis (A), harvested for whole extracts, prior to Western blots for the indicated proteins (B), used for Masson’s trichrome staining to determine collagen I expression (C), or processed for immunohistochemistry with double-staining for TM4SF5 and CD151 (D). (E) Human normal or liver tumor tissues were processed for Western blots or immunohistochemistry to identify CD151 (top), TM4SF5 (middle), and CD63 (bottom) and the nuclei were stained using DAPI. Scale bars depict 10 µm. Data represent three independent experiments.

Article Snippet: Double-immunostaining of human liver tissues were obtained from each patient as reported previously after informed content , or murine liver tissues were incubated with primary antibodies specific for TM4SF5 (Clone # 27, anti-TM4SF5 mAb, see above), CD63, and CD151 (Cell Signaling Technol.) with or without DAPI staining.

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Expressing, Immunohistochemistry, Double Staining

Journal: PLoS ONE

Article Title: Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

doi: 10.1371/journal.pone.0102817

Figure Lengend Snippet: (A to C) TM4SF5-expressing Chang-TGFβ1 (A and B) or Huh7 cells (C) transfected with shRNA against TM4SF5 (shTM4SF5) or a control-scrambled sequence (shControl) were processed for RT-PCR (A) or Western blots (B and C) against the indicated molecules. (D to F) TM4SF5-null Chang (D and E) or SNU449 (F) cells transfected with TM4SF5 cDNA or control plasmids (Mock) were processed for RT-PCR (D) or Western Blots (E and F). Note that CD63 immunoblots showed multiple bands, presumably due to 3 possible isoforms with N- glycosylations ( http://www.uniprot.org/uniprot/P08962 ). Data represent three different experiments.

Article Snippet: Double-immunostaining of human liver tissues were obtained from each patient as reported previously after informed content , or murine liver tissues were incubated with primary antibodies specific for TM4SF5 (Clone # 27, anti-TM4SF5 mAb, see above), CD63, and CD151 (Cell Signaling Technol.) with or without DAPI staining.

Techniques: Expressing, Transfection, shRNA, Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

doi: 10.1371/journal.pone.0102817

Figure Lengend Snippet: (A and B) Chang-TGFβ1 cells (A) or Huh7 cells (B) transfected with CD63 cDNA or a control plasmid (Mock) were processed for RT-PCR or Western blots. (C) Chang cells transfected with pGL3-TM4SF5 promoter DNA without or with CD63 cDNA for 24 h were treated with vehicle or TGFβ1 for additional 24 h, prior to luciferase reporter gene assay. Each value was shown at mean ± standard deviation. * depicts statistical significance ( p <0.05) and ** depicts insignificance ( p ≥0.05). (D and E) Chang and Chang-TGFβ1 cells were immunostained for TM4SF5 (green, D), CD63 (green, E), or CD151 (red, E) in addition to nuclear staining using DAPI. (F) Chang-TGFβ1 cells were immunostained for TM4SF5 (green) and either CD151 (top panel) or CD63 (bottom panel) in addition to DAPI staining. White box depicts area for an enlarged insert. (G) Chang cells transiently transfected with FLAG-TM4SF5 or CD151 were immunostained for CD63 (blue or red) and either FLAG-TM4SF5 (green) or CD151 (green) prior to visualization by confocal microscopy. (H) Chang cells transfected with FLAG-TM4SF5 for 48 h were immunostained using anti-FLAG (green), anti-LAMP2 (a lysosomal marker, blue), and anti-CD63 (red) antibody, prior to visualizations using confocal microscopy. Scale bars depict 10 µm. Data represent three independent experiments.

Article Snippet: Double-immunostaining of human liver tissues were obtained from each patient as reported previously after informed content , or murine liver tissues were incubated with primary antibodies specific for TM4SF5 (Clone # 27, anti-TM4SF5 mAb, see above), CD63, and CD151 (Cell Signaling Technol.) with or without DAPI staining.

Techniques: Transfection, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Reporter Gene Assay, Standard Deviation, Staining, Confocal Microscopy, Marker

Journal: PLoS ONE

Article Title: Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

doi: 10.1371/journal.pone.0102817

Figure Lengend Snippet: (A) Whole cell lysates from Chang cells transfected with control Strep or Strep-TM4SF5 plasmid were immunoprecipitated (IP) with anti-streptavidin-coated agarose beads before immunoblotting using anti-CD151, anti-CD63, or anti-streptavidin. (B) HCC827 lung carcinoma cells transfected with FLAG-mock or FLAG-TM4SF5 were harvested for whole cell extracts. The extracts were immunoprecipitated (IP) with anti-CD151 antibody prior to immunoblotting for FLAG and CD151, in parallel with lysates. (C) Whole cells extracts from Chang-TGFβ1 cells were immunoprecipitated with normal (Nor.) IgG or anti-CD151 (α-CD151) antibody, prior to standard Western blots for CD151 or TM4SF5, in parallel with whole cell lysates (WCL). (D) Chang cells transfected with FLAG-TM4SF5 were immunostained for CD151 (red) and FLAG (green), prior to visualization using a confocal microscope. Note that some TM4SF5 co-localized with CD151 at the membrane surface and internally in endosomal regions, but other TM4SF5 localized independently of CD151 even on the membrane surface. The images were presented in an independent duplicate for the same condition. Data represent three independent experiments.

Article Snippet: Double-immunostaining of human liver tissues were obtained from each patient as reported previously after informed content , or murine liver tissues were incubated with primary antibodies specific for TM4SF5 (Clone # 27, anti-TM4SF5 mAb, see above), CD63, and CD151 (Cell Signaling Technol.) with or without DAPI staining.

Techniques: Transfection, Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Microscopy, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone

doi: 10.3389/fnmol.2015.00040

Figure Lengend Snippet: Canonical members of HSPB family prevent the aggregation of Aβ-GFP. Cells were transfected with HSPB1, HSPB5, HSPB7 or FRTTO at 1:3 ratio for 48 h. Quantification of the pellet/soluble ratio of Aβ-GFP relative to FRTTO was depicted in the chart above the blot. Values represent mean ± SE of two independent experiments. ** P < 0.01, ns = non significant.

Article Snippet: The membranes were blocked with 5% dry milk in PBS with 0.1% Tween 20 (PBST) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: 6E10 (1:1000 in TBST, Covance), anti V5 (1:5000 in PBST, Invitrogen), anti β-actin (1:1000 in PBST, Abcam), anti HSPB1 (1:1000 in PBST, Stress Marq Biosciences), anti HSPB5 (1:1000 in PBST, Stress Marq Biosciences) and anti HSPB7 (1:1000, Abnova).

Techniques: Transfection