rabbit polyclonal anti vamp4  (Proteintech)

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: The Journal of Biological Chemistry

Article Title: Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans -Golgi Network *

doi: 10.1074/jbc.M110.137414

Figure Lengend Snippet: Endogenous CAPS1 is associated with the Golgi membrane. A and B, electron micrograms of submicrosomal fractions affinity-purified using immune magnetic beads. Cerebellar microsomal fractions of P21 mice were incubated with magnetic beads coated with anti-CAPS1 antibody (A) or anti-VAMP2 antibody (B), and immunoaffinity-purified submicrosomal fractions were subjected to electron microscopic observation. Scale bars, 200 nm. C, Western blot analysis of submicrosomal fractions affinity-purified using normal rabbit IgG, anti-CAPS1 antibody, or anti-VAMP2 antibody immunomagnetic beads. Each immunopurified fraction was immunoblotted with anti-CAPS1, anti-VAMP2, anti-VAMP4, anti-SNAP25, anti-Bcl-2, anti-BiP/GRP78, anti-pan-ARF (1D9), anti-GM130, anti-GS28, anti-p115, anti-Stx5, anti-Stx6, anti-Stx16, and anti-Vti1a antibodies. Anti-pan-ARF (1D9) antibody recognizes all of the ARF family proteins (supplemental Fig. S3).

Article Snippet: The following primary antibodies were also used for Western blotting: rabbit polyclonal anti-CAPS2 (1:1,000 dilution) ( 5 ), mouse monoclonal anti-FLAG (1:1,000 dilution; catalog no. F1804, Sigma), rat monoclonal anti-hemagglutinin epitope (HA) (1:1,000 dilution; catalog no. 1867423, Roche Applied Science), mouse monoclonal anti-VAMP2 (1:10,000 dilution; catalog no. 104211, Synaptic Systems), rabbit polyclonal anti-VAMP4 (1:1,000 dilution; catalog no. PA1-768, Affinity BioReagents), mouse monoclonal SNAP25 (1:2,500 dilution; catalog no. SMI-81, Sternberger), mouse monoclonal anti-ARF (clone 1D9) (1:500 dilution; catalog no. ab2806, Abcam), mouse monoclonal anti-GM130 (1:250 dilution; catalog no. 610822, BD Biosciences), mouse monoclonal anti-GS28 (1:1,000 dilution; catalog no. 611184, BD Biosciences), mouse monoclonal anti-p115 (1:1,000 dilution; catalog no. 612260, BD Biosciences), mouse monoclonal anti-Bcl-2 (1:500 dilution; catalog no. 610538, BD Biosciences), mouse monoclonal anti-BiP/GRP78 (1:250 dilution; catalog no. 610978, BD Biosciences), rabbit polyclonal anti-syntaxin 5 (1:1,000 dilution; catalog no. 110053, Synaptic Systems), mouse monoclonal anti-syntaxin 6 (1:2,500 dilution; catalog no. 610635, BD Biosciences), rabbit polyclonal anti-syntaxin 16 (1:1,000 dilution; catalog no. 110162, Synaptic Systems), mouse monoclonal anti-Vti1a (1:2,500 dilution; catalog no. 611220, BD Biosciences), and goat polyclonal anti-GST (1:1,000 dilution; catalog no. 27-4577, Amersham Biosciences).

Techniques: Affinity Purification, Magnetic Beads, Incubation, Purification, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans -Golgi Network *

doi: 10.1074/jbc.M110.137414

Figure Lengend Snippet: The binding between the PH domain of CAPS1 and ARF5 induces the accumulation of GDP-locked ARF5 in the Golgi membrane. A–F, subcellular localization of the C-terminal EGFP-HA-tagged CAPS1(PH), the C-terminal FLAG-tagged GDP-locked ARF5(T31N), and endogenous VAMP4 in PC12 cells. A–C, immunostaining of CAPS1(PH)-EGFP-HA and ARF(T31N)-FLAG with anti-HA (A) and anti-FLAG (B) antibodies, respectively, and the merged image (C). D–F, immunostaining of ARF5(T31N)-FLAG and endogenous VAMP4 with anti-FLAG (D) and anti-VAMP4 (E) antibodies, respectively, and the merged image (F). Scale bars (C and F), 10 μm. G–I, subcellular localization of the C-terminal HA-tagged CAPS1(dPH) and the C-terminal FLAG-tagged ARF5(T31N) in PC12 cells. Immunostaining of CAPS1(dPH)-HA and ARF5(T31N)-FLAG with anti-HA (G) and anti-FLAG (H) antibodies, respectively, and the merged image (I). Scale bars, 10 μm. J–M, subcellular localization of endogenous ARF5 in PC12 cells transfected with EGFP together with either control (J and K) or CAPS1 siRNA (L and M). The arrows indicate EGFP-expressing siRNA-transfected cells. Shown is immunostaining of endogenous ARF5 with anti-ARF5 antibody (K and L). Scale bars (K and M), 10 μm. N, ARF5 immunoreactivity levels merged with immunoreactivity for the Golgi complex. PC12 cells were transfected with the Golgi marker B4galt1-tdTomato together with either control (white bar) or CAPS1 siRNA (black bar). The ratio of ARF5 immunoreactivity in the Golgi complex (merged with B4galt1-tdTomato fluorescence) and that in whole cell soma was qualified. **, p < 0.01 by Student's t test. Error bars, S.E. O and P, subcellular distribution of GTP-locked ARF5(Q71L)-FLAG (O) and GDP-locked ARF5(T31N)-FLAG (P) expressed in primary cultured mouse hippocampal neurons. Cultures were transfected at 6 days in vitro, fixed at 8 days in vitro, and immunostained with anti-FLAG antibody. ax, axon; de, dendrite. Scale bar, 50 μm.

Article Snippet: The following primary antibodies were also used for Western blotting: rabbit polyclonal anti-CAPS2 (1:1,000 dilution) ( 5 ), mouse monoclonal anti-FLAG (1:1,000 dilution; catalog no. F1804, Sigma), rat monoclonal anti-hemagglutinin epitope (HA) (1:1,000 dilution; catalog no. 1867423, Roche Applied Science), mouse monoclonal anti-VAMP2 (1:10,000 dilution; catalog no. 104211, Synaptic Systems), rabbit polyclonal anti-VAMP4 (1:1,000 dilution; catalog no. PA1-768, Affinity BioReagents), mouse monoclonal SNAP25 (1:2,500 dilution; catalog no. SMI-81, Sternberger), mouse monoclonal anti-ARF (clone 1D9) (1:500 dilution; catalog no. ab2806, Abcam), mouse monoclonal anti-GM130 (1:250 dilution; catalog no. 610822, BD Biosciences), mouse monoclonal anti-GS28 (1:1,000 dilution; catalog no. 611184, BD Biosciences), mouse monoclonal anti-p115 (1:1,000 dilution; catalog no. 612260, BD Biosciences), mouse monoclonal anti-Bcl-2 (1:500 dilution; catalog no. 610538, BD Biosciences), mouse monoclonal anti-BiP/GRP78 (1:250 dilution; catalog no. 610978, BD Biosciences), rabbit polyclonal anti-syntaxin 5 (1:1,000 dilution; catalog no. 110053, Synaptic Systems), mouse monoclonal anti-syntaxin 6 (1:2,500 dilution; catalog no. 610635, BD Biosciences), rabbit polyclonal anti-syntaxin 16 (1:1,000 dilution; catalog no. 110162, Synaptic Systems), mouse monoclonal anti-Vti1a (1:2,500 dilution; catalog no. 611220, BD Biosciences), and goat polyclonal anti-GST (1:1,000 dilution; catalog no. 27-4577, Amersham Biosciences).

Techniques: Binding Assay, Immunostaining, Transfection, Expressing, Marker, Fluorescence, Cell Culture, In Vitro