Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, SDS Page, Western Blot, Infection, Staining, Software, Construct, Expressing

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3+ signal on or proximal to (i.e. within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with Bap31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in (). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. ( E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from fractions 1-7 (representing disassembled virus) and fraction 8 (representing assembled virus) was quantified and normalized to the Scr control. ** p ≤ 0.01; ns= not significant.

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, Infection, Staining, Membrane, Transport Assay, SDS Page, Western Blot, Virus

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP+Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Construct, Staining, Infection, Expressing, Control, SDS Page, Western Blot

Journal: Nature Communications

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

doi: 10.1038/s41467-021-24817-y

Figure Lengend Snippet: a Quantification and exemplary images of a PLA between SARS-CoV-2 Spike and ACE2 in Calu-3 depleted of IFITM1, IFITM2, or IFITM3 and infected with genuine SARS-CoV-2. Bars represent the mean of four independent experiments (100 cells ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. b Quantification and exemplary images of a PLA between Spike and ACE2 in Calu-3 cells depleted of IFITM2 and infected with SARS-CoV-2 virus on ice for 2 h and then incubated for 15 min at 37 °C. Bars represent the mean of four independent experiments (300 cells, ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. c Quantification and exemplary images of a PLA assay between Spike and RAB5A as in ( c ). Bars represent the mean of four independent experiments (400 cells, ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. DAPI (blue), nuclei. PLA signal (yellow). Scale bar, 20 µm. d Summary of the quantification shown in panels ( b ) (Spike-ACE2) and ( c ) (Spike-RAB5α) proximity upon SARS-CoV-2 infection. Individual data points and statistics are provided in panels ( b ) and ( c ). e Exemplary images of the colocalization (white) of PLA foci (red) indicating SARS-CoV-2 Spike and IFITM2 with early (EEA1, green, upper panel) or late endosomal markers (Rab7a, green, lower panel) in Calu-3 cells. Cells were infected with SARS-CoV-2 for 2 h at 4 °C or 2 h at 4 °C and 15 min at 37 °C as indicated. DAPI (blue), nuclei. f Quantification of the percentage of colocalization between the PLA signal and the indicated endosomal markers in 225 × 225 µm images. Bars represent the mean of four individual images (±SEM), unpaired t test, ** p = 0.0031. g Quantification of the combined PLA (IFITM2/Spike) in ( e ). Bars represent the mean of 120 cells (±SEM) over two independent experiments. The experiment was replicated twice to similar results. Two-sided Wilcoxon matched-pairs test, **** p < 0.0001. a – c , e Scale bars, 20 μm.

Article Snippet: For staining following antibodies were used: IFITM1 (α-IFITM1 Cell Signaling 13126S), IFITM2 (α-IFITM2 Abcam 236735), IFITM3 (α-IFITM3 Cell Signaling 59212S), SARS Spike CoV-2 (SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9], GTX-GTX632604), Rab5 alpha (Rab5 (RAB5A) Goat Polyclonal Antibody Origene AB0009-200) and ACE2 (Rabbit polyclonal anti-ACE2 Abcam, ab166755).

Techniques: Infection, Virus, Incubation

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion does not affect Rab5 recruitment to the endosomes and ligand-bound EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab5 and EEA1 (A), Rab5 and CD63 (B), and Rab5 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Right (A–C): single Z-plane of images. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab5 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab5 staining quantified in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Scale bars: 10 μm. p values were not statistically significant. Zoomed images correspond to the dashed inset boxes of the indicated images. Note: (B) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (B) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (B) and (C) is intentional.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 1. Effects of 3A5C7 monoclonal antibody (mAb) on morphine-induced mu-opioid receptor (MOR) endocytosis from cell membrane to cytoplasm. (A) Immunofluorescence staining indicated the endocytosis of MOR in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (B) Immunofluorescence staining indicated the endocytosis of MOR in SH- SY5Y cells treated with morphine and 3A5C7 mAb. (C) Immunofluorescence staining manifested the colocalization of MOR and Rab5 in HEK293T-MOR cells subjected to 3A5C7 mAb and morphine. (D, E) Flow cytometry showed the endocytosis of MOR in HEK293T-MOR cells co-treated with morphine and 3A5C7 mAb. (F, G) Flow cytometry showed the

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Membrane, Staining, Flow Cytometry

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 6. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance and physical dependence in mice. (A) Western blots indicating the cross specificity of 3A5C7 mAb against mu-opioid receptor (MOR) in the cortex, cerebellum, and hippocampus from C57/B6 mice. (B) Experiment flowchart for testing the effects of chronically administered 3A5C7 mAb on morphine tolerance and dependence. (C) The effects of chronically administered 3A5C7 mAb on the antinociceptive effects of morphine in mice measured by hotplate test. (D) The body weight changes of morphine-tolerant mice. Two-way analysis of variance with Bonferroni's post hoc tests were used for statistical analysis (n ¼ 6e7 mice in each group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, morphine þ mAb group vs. morphine group. #P < 0.05, ##P < 0.01, and ###P < 0.001, morphine þ mAb group vs. morphine þ normal IgG (NIg) group. (E) Representative immunofluorescence staining of MOR and Rab5 in the mouse dorsal root ganglions (DRGs) after

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Western Blot, Staining

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 7. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance via G protein-coupled receptor kinase 2 (GRK2)/b-arrestin2 pathway in mice. (A) Immunoblot showing that GRK2 was knocked-down by short hairpin ribonucleic acid (shRNA) in dorsal root ganglions (DRGs). (B) Quantification of the protein level of GRK2 in Fig. 7A. (C) Immunoblot showing that b-arrestin2 was knocked-down by shRNA in DRGs. (D) Quantification of the protein level of b-arrestin2 in Fig. 7C. (E) Hotplate tests demonstrating the effects of GRK2 or b-arrestin2 knockdown on the anti-tolerance efficacy of mAb 3A5C7. Two-way analysis of variance (ANOVA) with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ****P < 0.0001, sh-NC þ morphine þ mAb group vs. sh-NC þ morphine group; ###P < 0.001 and ####P < 0.0001, sh- NC þ morphine þ mAb group vs. sh-GRK2 þ morphine þ mAb group; &P < 0.05 and &&&P < 0.001, sh-NC þ morphine þ mAb group vs. sh-b-arrestin2 þ morphine þ mAb group (n ¼ 5 mice in each group). (F) Representative immunofluorescence images of MOR and Rab5 for each treatment in mouse DRGs (n ¼ 3 mice in each group). (G) The protein levels of hippocampal protein kinase A (PKA) from mice in each treatment group. (H, I) Quantification of the relative protein levels of PKA from Fig. 7G. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ***P < 0.001. (J) Inhibitory effects of acute 3A5C7 mAb administration on naloxone-precipitated withdrawal jumping were reduced in GRK2- and b-arrestin2-knockdown mice. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. ****P < 0.0001 vs. sh-NC þ morphine group; ####P < 0.0001 vs. sh-NC þ morphine þ mAb group (n ¼ 5 mice in each group). Data were presented as the mean ± standard error of mean. sh-NC: control shRNA; sh-GRK2: shRNA for GRK2; sh-b-arrestin2: shRNA for b-arrestin2; MPE: maximum possible effect; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Western Blot, shRNA, Knockdown, Control

Journal: Bioactive Materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Substrate stiffness-induced DDR1 activation is independent of collagen binding. (A) to (C) Western blot assay to determine phosphorylation of DDR1 at tyrosine 792 (p-DDR1) and the total DDR1 expression. GAPDH served as an internal control. Shown are representative blots from at least 3 independent experiments. Semi-quantification (in the lower panels) of indicated protein was performed by using the ImageJ software based on the analysis of the gray band intensity. (A) The p-DDR1 and total DDR1 levels in SMCs cultured for 24 h on collagen type I (Col) coated-polyacrylamide (PA) gels with a stiffness of 2 (soft) or 20 (stiff) kPa. (B) The p-DDR1 levels in SMCs grown on Col-coated gels for 24 h in the presence of the DDR1: Fc peptides. (C) The p-DDR1 and total DDR1 levels in SMCs cultured for 24 h on fibronectin (Fn)-coated PA gels. (D) Representative stimulated emission depletion microscopy (STED) images of DDR1 and Rab5A immunofluorescence in SMCs on Fn-coated gels. Cells were either pretreated with DDR1-IN-1 or DMSO. (E) Schematic diagram illustrating measuring the interaction between the collagen and the surfaces of SMCs by atomic force microscopy (AFM). (F) AFM to determine the adhesion probability and rupture forces between the Col- or BSA-coated probe and the cell surface. Each dot represents one cell. (G) and (H) AFM to determine the adhesion probability and rupture forces between the DDR1 ligand (Col) and the cell surface. In (G) , each dot represents an independent experiment, and in (H) , each dot represents the rupture force of a single bond between Col and the cell surface. (I) AFM to determine the adhesion probability in cells that were pretreated with DDR1-IN-1 or DMSO and cultured on Fn-coated gels. Each dot represents an independent experiment. (J) Western blot assay to determine DDR1 and GAPDH in cell lysates prepared using a non-reducing condition. (K) Quantitative RT-PCR to determine the expressions of DDR1 targeted genes in SMCs on Fn-coated gels. Each dot represents an independent experiment. * P < 0.05 vs. the indicated group.

Article Snippet: Rabbit polyclonal antibodies against DNMT1, p53, and DDR1, and mouse monoclonal antibody against Rab5A were purchased from Proteintech.

Techniques: Activation Assay, Binding Assay, Western Blot, Phospho-proteomics, Expressing, Control, Software, Cell Culture, Microscopy, Immunofluorescence, Quantitative RT-PCR

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Schematic of transposon insertion into exon 3 of Pclo genomic sequence creating a translational stop codon, truncating the generation of long Piccolo isoforms. ( B ) PCR verification of genotypes. PCR from Pclo wt/wt , Pclo wt/gt and Pclo gt/gt genomic DNA results in the amplification of 260 bp WT and/or 450 bp KO specific bands, respectively. ( C and D ) Western blot analysis of P0-P2 brain lysates ( C ) and lysates from DIV14 cultured hippocampal neurons ( D ). Bands, corresponding to Piccolo isoforms (70–560 kDa), are detected in Pclo wt/wt and Pclo wt/gt brain lysates as well as Pclo wt/wt neuron lysates, but not in Pclo gt/gt lysates (n = 3 independent experiments). ( E ) Immunocytochemical staining of hippocampal neurons. Piccolo immuno-reactivity co-localizes with VGlut1 in Pclo wt/wt neurons, but not in Pclo gt/gt neurons. Right panel, quantification of Piccolo intensities at VGlut1 puncta ( Pclo wt/wt = 1 ± 0.03, n = 586 synapses; Pclo gt/gt = 0.35 ± 0.01, n = 718 synapses; two independent experiments). ( F ) Immuno-histological staining of Pclo wt/wt and Pclo gt/gt brain sections. Representative images from the CA1 region of the hippocampus are shown. No Piccolo immuno-reactivity can be observed on Pclo gt/gt sections. ( G ) Western blot analysis of P0-P2 brain lysates. Expression levels of synaptic proteins are altered in Pclo gt/gt brain lysates (EEA1: Pclo wt/wt = 1 ± 0.03, Pclo wt/gt = 0.44 ± 0.01, Pclo gt/gt = 0.50 ± 0.02; two independent experiments; Rab7: Pclo wt/wt = 1 ± 0.08, Pclo wt/gt = 1.23 ± 0.05, Pclo gt/gt = 0.83 ± 0.13; four independent experiments; Rab5: Pclo wt/wt = 1 ± 0.09, Pclo wt/gt = 1.04 ± 0.11, Pclo gt/gt = 1.22 ± 0.04; three independent experiments; Bassoon: Pclo wt/wt = 1 ± 0.07, Pclo wt/gt = 0.95 ± 0.16, Pclo gt/gt = 0.80 ± 0.16; four independent experiments; Synaptophysin: Pclo wt/wt = 1 ± 0.11, Pclo wt/gt = 0.86 ± 0.13, Pclo gt/gt = 0.82 ± 0.21; three independent experiments; VGlut1: Pclo wt/wt = 1 ± 0.05, Pclo wt/gt = 1.05 ± 0.15, Pclo gt/gt = 0.83 ± 0.12; three independent experiments; Dynamin: Pclo wt/wt = 1 ± 0.11, Pclo wt/gt = 1.44 ± 0.16, Pclo gt/gt = 1.17 ± 0.05; three independent experiments). ( H ) Immunocytochemical staining of hippocampal neurons with antibodies against Homer, VGlut1 and MAP2 reveal the presence of VGlut1/Homer positive synapses along MAP2 positive dendrites in Pclo wt/wt and Pclo gt/gt neurons. ( I ) Images, of hippocampal neurons immunostained with antibodies against Synapsin and MAP2. The number of Synapsin positive puncta along primary dendrites (MAP2) does not differ between Pclo wt/wt and Pclo gt/gt neurons (left panel, Pclo wt/wt = 0.27 ± 0.02, n = 27 primary dendrites; Pclo gt/gt = 0.29 ± 0.02, n = 22 primary dendrites; two independent experiments). Scale bar in E, H and I 10 μm, scale bar in F 50 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Students T-test. **** denotes p<0.0001. 10.7554/eLife.46629.004 Figure 1—source data 1. This spreadsheet contains the normalized values used to generate the bar plots shown in .

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Sequencing, Amplification, Western Blot, Cell Culture, Staining, Expressing

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Schematic of SV membrane trafficking, illustrating when Rab5, EEA1 and Rab7 become associated with endosomal membranes. ( B ) Images of axon segments expressing GFP-Rab5 or GFP-Rab7 or immuno-stained with antibodies against EEA1. ( C ) Quantification of B. In Pclo wt/wt neurons, fewer GFP-Rab5 puncta are present per unit length of axon ( Pclo wt/wt = 0.06 ± 0.004, n = 139 axon sections; Pclo gt/gt = 0.08 ± 0.004, n = 143 axon sections; three independent experiments). In Pclo wt/wt neurons, more GFP-Rab7 puncta are present per unit length of axon ( Pclo wt/wt = 0.11 ± 0.004, n = 139 axon sections; Pclo gt/gt = 0.06 ± 0.004, n = 142 axon sections; two independent experiments). In Pclo wt/wt neurons, more EEA1 positive puncta are present per unit length of axon compared to Pclo gt/gt neurons ( Pclo wt/wt = 0.12 ± 0.01, n = 29 axon sections; Pclo gt/gt = 0.09 ± 0.004, n = 34 axon sections; two independent experiments). ( D–F ) Immunocytochemical stainings of hippocampal neurons for endosome proteins. ( D ) Rab5 is present at Pclo wt/wt and Pclo gt/gt synapses, no differences in intensities are detectable. ( E ) EEA1 intensities are significantly reduced in Pclo gt/gt synapses. ( F ) mChRab7 intensities are significantly reduced in Pclo gt/gt synapses. ( G–I ) Quantification of ( D–F ). ( G ) The levels of Rab5 are not altered between Pclo wt/wt and Pclo gt/gt synapses ( Pclo wt/wt = 1 ± 0.03, n = 808 synapses; Pclo gt/gt = 1.04 ± 0.05, n = 773 synapses; three independent experiments). ( H ) EEA1 intensity is significantly reduced in Pclo gt/gt synapses ( Pclo wt/wt = 1 ± 0.02, n = 4323 synapses; Pclo gt/gt = 0.79 ± 0.02, n = 3939 synapses; 10 independent experiments). ( I ) mChRab7 intensity is reduced in Pclo gt/gt synapses ( Pclo wt/wt = 1 ± 0.03, n = 386 synapses; Pclo gt/gt = 0.72 ± 0.02, n = 525 synapses; three independent experiments). Scale bar represents 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t -test. * denotes p<0.05, *** denotes p<0.001 and **** denotes p<0.0001. 10.7554/eLife.46629.013 Figure 4—source data 1. This spreadsheet contains the normalized values used to generate the bar plots shown in .

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Expressing, Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) GFP-2x-FYVE was used as a reporter for PI3P. In HEK293 cells, GFP-2x-FYVE decorates single vesicular early endosomes. ( B ) Lentiviral expression of GFP-2x-FYVE in primary hippocampal Pclo wt/wt and Pclo gt/gt neurons decorates small organelles along axons indicating the presence of PI3P. ( C ) Quantification of ( B ). The number of GFP-2x-FYVE-positive organelles along axons is not altered in Pclo gt/gt compared to Pclo wt/wt neurons ( Pclo wt/wt = 0.88 ± 0.05, n = 39 axons; Pclo gt/gt = 0.80 ± 0.05; three independent experiments). ( D ) Pclo wt/wt neurons expressing GFP-2x-FYVE stained for Synaptophysin. Many of GFP-2x-FYVE positive puncta along axons co-localize with synapses marked by Synaptophysin. ( E ) Quantification of ( D ). 65.88 ± 5.46% of GFP-2x-FYVE-positive puncta co-localize with Synaptophysin. ( F ) Images representing GFP-2x-FYVE puncta along axons in Pclo wt/w t as well as Pclo gt/gt neurons. GFP-2x-FYVE intensity is slightly but not significantly increased in Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.02, n = 1516 synapses; Pclo gt/gt = 1.03 ± 0.02, n = 1137 synapses; 11 independent experiments). ( G ) Super resolution structural illumination microscopy (SIM) of Pclo wt/wt and Pclo gt/gt neurons expressing GFP-2x-FYVE. Synaptic vesicle clusters marked with Synaptophysin (red) can be spatially separated from the active zone protein Bassoon (blue). The PI3P-positive compartment lies above the active zone within the vesicle cluster. The size of GFP-2x-FYVE organelles appears smaller in Pclo gt/gt terminals compared to Pclo wt/wt terminals. ( H ) Histogram of the area of the measured GFP-2x-FYVE compartments reveals that the size of GFP-2x-FYVE organelles, in Pclo gt/gt neurons, is shifted towards smaller areas (n = 2 independent experiments). ( I ) % co-localization of Rab5 positive puncta with Synaptophysin. 83,56 ± 4.67% of Rab5 positive puncta co-localize with Synaptophysin (three independent experiments). Scale bar in A, B and D represents 10 μm. Scale bar in F represents 500 nm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t-test.

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Expressing, Staining, Microscopy

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: Cultured hippocampal Pclo wt/wt or Pclo gt/gt neurons were stained for different endosome proteins. Subsequently, their intensity was measured in the cell soma ( A–D ) or along dendrites ( E–H ) marked by MAP2. No difference in the staining intensity of GFP-2x-FYVE ( A ), EEA1 ( B ) or Rab5 ( C ) is detectable at the somata of Pclo wt/wt vs Pclo gt/gt neurons (GFP-2x-FYVE: Pclo wt/wt = 1 ± 0.08, n = 16 soma; Pclo gt/gt = 1.14 ± 0.32, n = 10 soma; three independent experiments; EEA1: Pclo wt/wt = 1 ± 0.05, n = 34 soma; Pclo gt/gt = 0.94 ± 0.16, n = 19 soma; p=0.6559; five independent experiments; Rab5: Pclo wt/wt = 1 ± 0.07, n = 18 soma; Pclo gt/gt = 0.92 ± 0.06, n = 18 soma; p=0.4158; three independent experiments). ( D ) Rabex5 intensity is decreased at somata of Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.07, n = 16 soma; Pclo gt/gt = 0.65 ± 0.06, n = 15 soma; two independent experiments). ( E ) Intensity of GFP-2x-FYVE (GFP) organelles along MAP2 positive dendrites is not different between Pclo wt/wt and Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.02, n = 451 puncta; Pclo gt/gt = 0.94 ± 0.02, n = 400; two independent experiments). ( F ) In Pclo gt/gt neurons, the intensity of EEA1-positive organelles along MAP2 positive dendrites is increased ( Pclo wt/wt = 1 ± 0.03, n = 462 puncta; Pclo gt/gt = 1.28 ± 0.04, n = 358; two independent experiments). ( G and H ) Intensity of Rab5 ( G ) along Pclo gt/gt dendrites is increased compared to Pclo wt/wt dendrites, however intensity of Rabex5 ( H ) is not different (Rab5: Pclo wt/wt = 1 ± 0.02, n = 405 puncta; Pclo gt/gt = 1.43 ± 0.05, n = 372; three independent experiments; Rabex5: Pclo wt/wt = 1 ± 0.03, n = 232 puncta; Pclo gt/gt = 1.06 ± 0.04, n = 278; two independent experiments). Scale bar represents 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM. Student`s t –test. *** denotes p<0.001.

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Cell Culture, Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Schematic of early endocytic trafficking steps. After pinching off from the plasma membrane early endocytic vesicles undergo consecutive maturation steps. The lipid PI3P is generated and a stable complex consisting of Rab5 and its GEF Rabex5 is formed creating a pool of active Rab5. This step is necessary to recruit EEA1 and form early endosomes. ( B ) Images depicting Rab5 and EEA1 intensities at GFP-2x-FYVE organelles along axons in Pclo gt/gt vs Pclo wt/wt neurons. ( C–F ) Quantification of B. ( C ) The levels of Rab5 at PI3P-positive organelles are decreased ( Pclo wt/wt = 1 ± 0.02, n = 1645 puncta; Pclo gt/gt = 0.81 ± 0.02, n = 1233 puncta; six independent experiments). ( D ) The amount of EEA1 at endosome membranes is reduced ( Pclo wt/wt = 1 ± 0.04, n = 1634 puncta; Pclo gt/gt = 0.37 ± 0.02, n = 1169 puncta; six independent experiments). ( E ) Quantification of double positive compartments along axons. The fraction of GFP-2x-FYVE/Rab5 is not altered ( Pclo gt/gt = 0.97 ± 0.33, n = 5 independent experiments). ( F ) The relative percentage of GFP-2x-FYVE/Rab5/EEA1 positive vesicles is decreased in Pclo gt/gt neurons (GFP-2x-FYVE/Rab5/EEA1: Pclo gt/gt = 0.28 ± 0.10, n = 6 independent experiments). Scale bars represent 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t -test. * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001 and **** denotes p<0.0001. 10.7554/eLife.46629.016 Figure 5—source data 1. This spreadsheet contains the normalized values used to generate the bar plots shown in .

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Generated

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Immunocytochemical staining of hippocampal neurons for Rabex5. ( B ) Quantification of ( A ). Rabex5 is present at Pclo wt/wt and Pclo gt/gt synapses, no difference in the intensities is detectable ( Pclo wt/wt = 1 ± 0.02, n = 2397 synapses; Pclo gt/gt = 0.96 ± 0.02, n = 1802 synapses; seven independent experiments). ( C ) Images depicting Rabex5 intensities at PI3P-positive organelles. ( D ) Quantification of ( C ). Less Rabex5 is present at PI3P-positive membranes in Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.04, n = 728 puncta; Pclo gt/gt = 0.80 ± 0.03, n = 652 puncta; six independent experiments). ( E ) Quantification of double positive compartments along axons. The fraction of GFP-2x-FYVE/Rabex5 double positive vesicles is increased in Pclo gt/gt neurons ( Pclo gt/gt = 1.49 ± 0.33, n = 5 independent experiments). ( F ) The relative percentage of GFP-2x-FYVE-Rabex5-Rab5 triple positive compartments is decreased in Pclo gt/gt neurons ( Pclo gt/gt = 0.57 ± 0.14, n = 5 independent experiments). Scale bar represents 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t-test.

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Representative images showing FM1-43 levels in Pclo wt/wt and Pclo gt/gt boutons either positive (arrow) or negative (arrowhead) for mCh-Rab5 Q79L after 60 mM KCl stimulation. ( B ) Quantification of ( A ). ( Pclo wt/wt = 1 ± 0.01, n = 528 puncta; Pclo gt/gt = 0.76 ± 0.01, n = 654 puncta; Pclo wt/wt (mCh-Rab5 Q79L ) =1.04 ± 0.02, n = 304 puncta; Pclo gt/gt (mCh-Rab5 Q79L ) =1.22 ± 0.02, n = 337 puncta; three independent experiments). ( C ) Left: Images depicting FM1-43 intensities in Pclo wt/wt and Pclo gt/gt boutons either positive (arrow) or negative (arrowhead) for mCh-Rab5 Q79L . Right: Images depicting FM1-43 unloading kinetics in Pclo wt/wt and Pclo gt/gt boutons either positive (arrow) or negative (arrowhead) for mCh-Rab5 Q79L . ( D ) Quantification of ( C ). The presence of mCh-Rab5 Q79L is not sufficient to rescue slowed FM1-43 unloading kinetics and total amount of FM1-43 dye released in Pclo gt/gt boutons ( Pclo gt/gt = 47.24 ± 0.68, n = 361 synapses; Pclo gt/gt (Rab5 Q79L ) =48.33 ± 0.53, n = 479 synapses; four independent experiments). In Pclo wt/wt boutons expressing mCh-Rab5 Q79L , FM1-43 unloading kinetics are slowed and total amounts of dye released are reduced similar to what is observed in Pclo gt/gt boutons ( Pclo wt/wt = 56.28 ± 0.81, n = 301 synapses; Pclo wt/wt (Rab5 Q79L ) =50.15 ± 0.85, n = 342 synapses; four independent experiments). Scale bars represent 5 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM. ANOVA with Tukey multi comparison test. * denotes p<0.05, **** denotes p<0.0001. 10.7554/eLife.46629.024 Figure 8—source data 1. This spreadsheet contains the normalized values used to generate the bar plots shown in .

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Expressing

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A and B ) Images of Pclo wt/wt ( A ) and Pclo gt/gt ( B ) neurons stained for Synaptophysin and Rab5 with and without TTX treatment. ( C ) Quantification of ( A and B ). Synaptic Rab5 levels do not significantly alter in Pclo wt/wt and Pclo gt/gt synapses upon TTX treatment ( Pclo wt/wt = 1 ± 0.05, n = 391 synapses; Pclo wt/wt (TTX) = 0.73 ± 0.05, n = 482 synapses; Pclo gt/gt = 1.14 ± 0.09, n = 400 synapses; Pclo gt/gt (TTX) = 0.90 ± 0.04, n = 372 synapses; two independent experiments). ( D and E ) Images of Pclo wt/wt ( D ) and Pclo gt/gt ( E ) neurons stained for Synaptophysin and EEA1 with and without TTX treatment. ( F ) Quantification of ( D and E ). In Pclo wt/wt synapses EEA1 levels drop upon TTX treatment ( Pclo wt/wt = 1 ± 0.03, n = 1653 synapse; Pclo wt/wt (TTX) = 0.72 ± 0.03, n = 1887 synapses; four independent experiments). In contrast, EEA1 levels slightly increase in Pclo gt/gt synapses due to TTX treatment ( Pclo gt/gt = 0.68 ± 0.04, n = 918; Pclo gt/gt (TTX) = 0.78 ± 0.03, n = 1352; four independent experiments). ( G and H ) Pclo wt/wt ( G ) and Pclo gt/gt ( H ) neurons stained for Synaptophysin and Rabex5 with and without TTX treatment. ( I ) Quantification of ( G and H ). Rabex5 level at synapses slightly decrease in Pclo wt/wt and Pclo gt/gt neurons upon TTX treatment ( Pclo wt/wt = 1 ± 0.02, n = 2124 puncta; Pclo wt/wt (TTX) = 0.85 ± 0.02, n = 2058 puncta; Pclo gt/gt = 0.93 ± 0.02, n = 1889, Pclo gt/gt (TTX) = 0.77 ± 0.02, n = 2305; six independent experiments). Scale bars represent 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, ANOVA with Tukey multi comparison test. * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001 and **** denotes p<0.0001.

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Pclo wt/wt neurons expressing GFP-2x-FYVE were treated for 30 min with Latrunculin or DMSO as control, fixed and stained for Rab5 and EEA1. Latrunculin treatment leads to increased levels of Rab5 at PI3P-positive organelles ( Pclo wt/wt - Latrunculin = 1 ± 0.03, n = 518 puncta; Pclo wt/wt + Latrunculin = 1.21±0.04, n = 579; three independent experiments). EEA1 levels at endosome membranes are not affected by Latrunculin treatment ( Pclo wt/wt - Latrunculin = 1 ± 0.04, n = 721 puncta; Pclo wt/wt + Latrunculin = 0.91±0.03, n = 535; three independent experiments). ( B ) Pclo gt/gt neurons expressing GFP-2x-FYVE were treated for 30 min with Jasplakinolide or DMSO as control, fixed and stained for Rab5 and EEA1. Jasplakinolide treatment does not alter Rab5 levels at PI3P-positive organelles. ( Pclo gt/gt - Jasplakinolide = 0.84 ± 0.04, n = 441 puncta; Pclo gt/gt + Jasplakinolide = 0.78±0.04, n = 377 puncta; three independent experiments). EEA1 levels at PI3P-positive organelles are also not altered through the treatment with Jasplakinolide ( Pclo gt/gt - Jasplakinolide = 0.66 ± 0.03, n = 601 puncta; Pclo gt/gt + Jasplakinolide = 0.67±0.04, n = 493 puncta; three independent experiments). Scale bar represents 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t-test. * denotes p<0.05, *** denotes p<0.001 and **** denotes p<0.0001.

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Expressing, Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: Piccolo (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:1000, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ), Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:1000; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), Bassoon (1:1000; rabbit; synaptic systems, Göttingen, Germany; Cat# 141002, RRID: AB_887698 ), Dynamin (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab52611, RRID: AB_869531 ).

Techniques: Recombinant, Subcloning, Plasmid Preparation, Sequencing