rabbit anti-vamp4 synaptic systems cat# 136002  (Synaptic Systems)

Journal: bioRxiv

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1101/2024.05.16.594502

Figure Lengend Snippet: a , Heatmap in which the Log 2 Fold Change across time points of axonal synaptic proteins and members of the SNARE complex is plotted. b , Live neuron expressing RUSH-LAMP2A-mNG, RUSH-SYT1-Halo and scramble, imaged during 1h of biotin addition. Still images show part of the Golgi and a budding event. Intensity profile graph in the bottom. c , Neurons expressing RUSH-LAMP2A-mNG and RUSH-SYT1-Halo at 1 and 4h post-release. Kymographs from live cell imaging along the axon every 1s for 180 s are shown. Colocalized anterograde (blue), retrograde (orange) or stationary (grey) trajectories were traced on the right. d , Quantification of the number of trajectories for 1 and 4h. n = 15 and 19 neurons; N=3. e , Confocal images of neurons expressing RUSH-LAMP1-V5 and EGFP-VAMP4, 1h after release. Blue and orange boxes indicate magnified areas shown on the right, with corresponding intensity profile graph. f - g , Confocal images of neurons expressing RUSH-LAMP1-V5 ( f ) or RUSH-Syt1-mNG ( g ) plus shRNA against VAMP4, or scramble. Quantifications of the number of RUSH-LAMP1 (n= 19 and 27 neurons; N=4) and SYT1 (n= 27 and 22 cells; N=3) positive compartments are shown on the right. h , Still images from the soma of neuron expressing RUSH-LAMP2a-mNG, RUSH-SYT1-Halo and shRNA against VAMP4; control scramble in (b). Images show a Golgi budding event after 1h release. Corresponding intensity profile graph on the right. i ) Neurons expressing RUSH-LAMP2A-mNG and shRNA against VAMP4 or scramble. Neurons were fixed after 1h release and stained for LAMTOR4. Magnified images from , and respective intensity profile graphs are shown. j ) Neurons transfected as in (h) were labeled for SirLyso and imaged live after 1h release. Still images from time points indicated in images and respective intensity profile graphs are shown. k ) Temporal intensity profile graph for RUSH-SYT1 and SirLyso from image in (j). See also & . Data are presented as mean values ± SD, plus individual points. ns–not significant; *p<0.05; *** p<0.001; **** p<0.0001. Mann-Whitney test in (d), (f), and (g).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-LAMTOR4 (Cell Signaling, clone D6A4V, Cat# 12284S, RRID: AB_2797870, 1/500), mouse anti-Stx6 (BD Biosciences Cat# 610635, RRID:AB_397965, 1/100), rabbit anti-VAMP4 (Synaptic Systems, Cat# 136002, RRID:AB_887816, 1/100), mouse anti-V5 (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564, 1/1000 for IF and WB), mouse anti-Pan-Neurofascin external (clone A12/18; UC Davis/NIH NeuroMab, Cat# 75-172, RRID: AB_2282826, 0.18 mg/ml), in-house rabbit anti-TRIM46 (1/1000), mouse anti-VTI1B (BD Biosciences Cat# 611404, RRID:AB_398926, 1/250), rabbit anti-GM130 (Abcam Cat# ab52649, RRID:AB_880266, 1/800).

Techniques: Expressing, Live Cell Imaging, shRNA, Staining, Transfection, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1101/2024.05.16.594502

Figure Lengend Snippet: a , Confocal images of somas from neurons expressing RUSH-LAMP1-V5 and scramble or VAMP4 shRNA, fixed 1h after release and stained for LAMTOR4 and GM130. c , Still images of the soma of a live neuron expressing RUSH-LAMP2A-mNG and RUSH-SYT1-Halo, and VAMP4 shRNA, 1h after release and labeled with SirLyso. Scale bar, 5 µm in (a) and (b).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-LAMTOR4 (Cell Signaling, clone D6A4V, Cat# 12284S, RRID: AB_2797870, 1/500), mouse anti-Stx6 (BD Biosciences Cat# 610635, RRID:AB_397965, 1/100), rabbit anti-VAMP4 (Synaptic Systems, Cat# 136002, RRID:AB_887816, 1/100), mouse anti-V5 (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564, 1/1000 for IF and WB), mouse anti-Pan-Neurofascin external (clone A12/18; UC Davis/NIH NeuroMab, Cat# 75-172, RRID: AB_2282826, 0.18 mg/ml), in-house rabbit anti-TRIM46 (1/1000), mouse anti-VTI1B (BD Biosciences Cat# 611404, RRID:AB_398926, 1/250), rabbit anti-GM130 (Abcam Cat# ab52649, RRID:AB_880266, 1/800).

Techniques: Expressing, shRNA, Staining, Labeling

Journal: Nature Communications

Article Title: Prion protein conversion at two distinct cellular sites precedes fibrillisation

doi: 10.1038/s41467-023-43961-1

Figure Lengend Snippet: A – D Persistently prion-infected S7 cells were co-labelled with 6D11 and vesicular markers ( A ) Scg2, ( B ) Syn1 and ( C ) Vamp4 as well as with constitutively secreted Col4 ( D ). E SIM image of plasma membrane, triple-labelled with Syn1, 6D11 and 5B2. Magnified areas (a–c) are denoted by a dashed box in the first image. F Diagram of marker proteins used to map trafficking routes of 6D11-positive PrP d . G Levels of colocalisation between 6D11 and organelle/vesicular markers, expressed as Person correlation coefficients. Data from two independent experiments with at least 10 per protein are shown. For representative images and gene names we refer to Supplementary Fig. . H Neuronal depolarisation with 25 mM KCl led to rapid depletion of Syn1-positive vesicles in S7 cells. Cells were fixed at 5 min after incubation with KCl. Arrows in magnified areas denote synaptic vesicles. I Increased levels of co-labelled PrP d aggregates at the plasma membrane, following KCl-evoked depolarisation. Cells were fixed at 5 min after incubation with KCl. J Quantitative changes of co-labelled PrP d after depolarisation in dependence of the KCl concentration normalised to untreated cells from three independent experiments with at least 36 replicates per condition. For statistical analysis, ANOVA with Bonferroni correction for multiple comparisons was conducted. K Quantitative changes in surface PrP levels after KCl-evoked depolarisation in uninfected S7 versus prion-infected iS7 cells. Following fixation, cells were stained with anti-PrP antibody 8H4. Data from three independent experiments with at least 48 images per experiment were analysed. Data represent mean values ± SEM. Statistical significance was evaluated by Student’s t -test ( p < 0.01). Source data are provided as a Source Data file.

Article Snippet: Mouse monoclonal anti-PrP (8H4, Sigma, P0110), rat monoclonal Lamp1 (1D4B, Santa Cruz Biotechnology (SCB), sc-19992), mouse monoclonal anti-PrP (5B2, SCB, sc-47730, used at a 1:500 dilution), mouse monoclonal anti-PrP (AH6, SCB, sc-69896), mouse monoclonal anti-PrP (8B4, SCB, sc-47729), mouse monoclonal anti-Syp (7.2, Synaptic Systems, 101 011), rabbit polyclonal anti-Vamp4 (Synaptic Systems, 136 002), rabbit polyclonal anti-Chga (Synaptic Systems, 259 003), rabbit polyclonal anti-phospho FAK (Tyr925, Cell Signaling Technology, 3284), rabbit polyclonal anti-beta Cop (Thermo Fisher Scientific, PA1-061), rabbit polyclonal anti-Scg2 (Abcam, ab12241), mouse monoclonal anti-PrP (7D9, Abcam, ab14219), rabbit polyclonal anti Col4 (AbD Serotech, 2150-1470), rat monoclonal anti-CD29 (9EG7, BD Biosciences, 553715), mouse monoclonal anti-GM130 (35, BD Biosciences, 610822), mouse monoclonal anti-PrP (6D11, BioLegend, 808002), mouse monoclonal anti-PrP (Saf32, Cayman, 189720), rabbit monoclonal anti-PDI (C81H6, Cell Signaling Technology, 3501 S), rabbit monoclonal anti-Eea1 (C45B10, Cell Signaling Technology, 3288), rabbit polyclonal anti-Lc3a (Cell Signaling Technology, 4599), rabbit polyclonal anti-GFAP (DAKO, Z0334, used at a 1:10,000 dilution), Mouse monoclonal anti-PrP (3F4, Merck, MAB1562), mouse monoclonal anti-PrP (MAB5424, Merck, MAB5424), mouse monoclonal anti-myc, AF 488-conjugated (9E10, Merck, 16-308), rabbit polyclonal anti-Syp1 (Millipore, AB1543), mouse monoclonal anti-Snap25 (SP14, Millipore, MAB331), rabbit polyclonal anti-Gorasp2 (Grasp55) (Proteintech, 10598-1-AP), mouse monoclonal anti-PrP (AG4, TSE Resources Centre, TSE RC, RC 059), mouse monoclonal anti-PrP (GE8, TSE Resources Centre, TSE RC, RC 061), ICSM18 and ICSM35 (UCL Institute of Prion Diseases), chicken polyclonal anti-Map2 (Abcam, ab5392).

Techniques: Infection, Membrane, Marker, Incubation, Concentration Assay, Staining

Journal: bioRxiv

Article Title: Apoptotic Extracellular Vesicles (ApoEVs) Safeguard Liver Homeostasis and Regeneration via Assembling an ApoEV-Golgi Organelle

doi: 10.1101/2021.02.24.432630

Figure Lengend Snippet: (A) Representative immunofluorescent images show apoEV (labeled with CellMask™ Deep Red, red) surface expression of vesicle-localized-soluble N -ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs, green) VAMP3, V VAMP4 and Golgi SNARE of 15 kDa (GS15). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs. Scale bars = 25 μm. (B) Western blot analysis show expression of VAMPs in apoEVs compared to their source MSCs. (C) Western blot analysis show expression of VAMPs in apoEVs. Source MSCs were transfected with small interferon RNAs (siRNAs) as a negative control (siCtrl) for VAMP3 (si Vamp3 ), VAMP4 (si Vamp4 ), and both VAMP3 and VAMP4 (si Vamp3/4 ). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs after knockdown of VAMPs. (D) Representative images show cultured Fas mutant ( Fas mut ) tetraploid hepatocytes (4N-HCs) with 130 kDa Golgi matrix protein (GM130) immunofluorescent staining (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). White dashed lines depict cell borders of cultured HCs. Imaging analysis was performed to quantify apoEV uptake and percentages of Golgi-contacted apoEVs. Scale bars = 10 μm. N = 4 per group. (E) Representative fluorescent images show cultured Fas mut 4N-HCs with ac-α-tubulin immunostaining (white), N -acetylgalactosaminyltransferase 2 (GALNT2)-GFP-labeled Golgi (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). Imaging analysis was performed to quantify HC percentages with Golgi fragmentation and acetylated microtubules (MTs) fluorescence intensity. Scale bars = 5 μm. N = 4 per group. (F) Western blot analysis showed human VAMP3 (hVAMP3) expression in isolated HC Golgi. ApoEVs were derived from human MSCs (hApoEVs). GM130 was used as an internal control. (G) Western blot analysis showed VAMP3 expression in apoEVs without or with treatment by 250 ng/ml Tetanus toxin (TeNT). (H) Representative Golgin84 immunofluorescent staining (green) images of liver Golgi counterstained with DAPI (blue). WT, wild type. Imaging analyses were performed to quantify HC percentages with Golgi fragmentation. Scale bars = 5 μm. N = 4 per group. (I) Golgi apparatus was isolated from the liver and Golgi protein mass was determined using the BCA method. N = 4 per group. (J) Representative liver fluorescent images showed HCs with different nuclei (blue, DAPI for DNA) and their cell borders (green, phalloidin for F-actin). # indicates binucleated HCs. Scale bars = 25 μm. N = 4 per group. (K) After PI staining, percentages of binucleated HCs were quantified. Diploid HCs (2N-HCs) were analyzed using flow cytometry. N = 4 per group. (L) Hematoxylin and eosin (H&E) staining of liver tissues in periportal vein (PV) areas. Hepatic injury scores were examined based on pathological parameters. Scale bars = 50 μm. N = 4 per group. (M) Serum alanine aminotransferase (ALT) levels were determined. N = 4 per group. (N) Graphical summary illustrating that apoEVs use VAMP3 to assemble with Golgi for HC and liver regulation. Data represent mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; NS, not significant, P > 0.05. Statistical analyses were performed by Student’s t test for two-group analysis or one-way analysis of variance followed by the Newman-Keuls post hoc tests for multiple group comparisons.

Article Snippet: For IF staining of surface markers of apoEVs, after isolation, apoEVs were stained with rabbit anti-human/mouse VAMP3 primary antibody (13640, Cell Signaling Technology, USA), rabbit anti-mouse VAMP4 primary antibody (PA1-768, Invitrogen, USA), mouse anti-mouse GS15 primary antibody (610960, BD Transduction Laboratories, UK), rabbit anti-mouse C1q primary antibody (ab71940, Abcam, UK), or mouse anti-mouse TSP1 primary antibody (sc-59887, Santa Cruz Biotechnology, USA) at 4°C for 1 h at a concentration of 1:100 in PBS.

Techniques: Labeling, Expressing, Staining, Western Blot, Transfection, Negative Control, Cell Culture, Mutagenesis, Imaging, Immunostaining, Fluorescence, Isolation, Derivative Assay, Flow Cytometry, Standard Deviation