Anti-Dynamin 1 antibody [EP801Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

Unpurified ab52611 at 1/100-1/250 dilution staining human brain tissue; paraffin embedded.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab52611 at a working dilution of 1/50. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

Immunohistochemical staining of paraffin embedded mouse cerebral cortex with purified ab52611 at a working dilution of 1/50. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• WB

Lab

Western blot - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Dynamin 1 antibody [EP801Y] (ab52611) at 1/10000 dilution

All lanes:

SH-SY5Y cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 97 kDa

Observed band size: 97 kDa

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• WB

Collaborator

Western blot - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

ab52611 was shown to react with DNM1 in wild-type U-2 OS cells in Western blot with loss of signal observed in a DNM1 knockout cell line. Wild-type U-2 OS and DNM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab52611 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2ug/mL before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Dynamin 1 antibody [EP801Y] (ab52611) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

DNM1 knockout U-2 OS cell lysate at 20 µg

Predicted band size: 97 kDa

false

• WB

Unknown

Western blot - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

All lanes:

Western blot - Anti-Dynamin 1 antibody [EP801Y] (ab52611) at 1/2000 dilution

All lanes:

3T3 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP antibody at 1/2000 dilution

Predicted band size: 84 kDa,97 kDa

Observed band size: 88 kDa,97 kDa

false

• WB

Lab

Western blot - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Dynamin 1 antibody [EP801Y] (ab52611) at 1/2000 dilution

Lane 1:

C6 cell lysate at 20 µg

Lane 2:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 97 kDa

Observed band size: 97 kDa

false

• WB

CiteAb

Western blot - Anti-Dynamin 1 antibody [EP801Y] (AB52611)

Dynamin 1 western blot using anti-Dynamin 1 antibody [EP801Y] ab52611. Publication image and figure legend from Ackermann, F., Schink, K. O., et al., 2019, Elife, PubMed 31074746.

ab52611 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab52611 please see the product overview.

Transposon mutagenesis leads to the loss of Piccolo protein in rats.(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 Pclowt/wt, Pclowt/gt and Pclogt/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 Pclowt/wt and Pclowt/gt brain lysates as well as Pclowt/wt neuron lysates, but not in Pclogt/gt lysates (n = 3 independent experiments). (E) Immunocytochemical staining of hippocampal neurons. Piccolo immuno-reactivity co-localizes with VGlut1 in Pclowt/wt neurons, but not in Pclogt/gt neurons. Right panel, quantification of Piccolo intensities at VGlut1 puncta (Pclowt/wt = 1 ± 0.03, n = 586 synapses; Pclogt/gt = 0.35 ± 0.01, n = 718 synapses; two independent experiments). (F) Immuno-histological staining of Pclowt/wt and Pclogt/gt brain sections. Representative images from the CA1 region of the hippocampus are shown. No Piccolo immuno-reactivity can be observed on Pclogt/gt sections. (G) Western blot analysis of P0-P2 brain lysates. Expression levels of synaptic proteins are altered in Pclogt/gt brain lysates (EEA1 : Pclowt/wt = 1 ± 0.03, Pclowt/gt = 0.44 ± 0.01, Pclogt/gt = 0.50 ± 0.02; two independent experiments; Rab7 : Pclowt/wt = 1 ± 0.08, Pclowt/gt = 1.23 ± 0.05, Pclogt/gt = 0.83 ± 0.13; four independent experiments; Rab5 : Pclowt/wt = 1 ± 0.09, Pclowt/gt = 1.04 ± 0.11, Pclogt/gt = 1.22 ± 0.04; three independent experiments; Bassoon : Pclowt/wt = 1 ± 0.07, Pclowt/gt = 0.95 ± 0.16, Pclogt/gt = 0.80 ± 0.16; four independent experiments; Synaptophysin : Pclowt/wt = 1 ± 0.11, Pclowt/gt = 0.86 ± 0.13, Pclogt/gt = 0.82 ± 0.21; three independent experiments; VGlut1 : Pclowt/wt = 1 ± 0.05, Pclowt/gt = 1.05 ± 0.15, Pclogt/gt = 0.83 ± 0.12; three independent experiments; Dynamin : Pclowt/wt = 1 ± 0.11, Pclowt/gt = 1.44 ± 0.16, Pclogt/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 Pclowt/wt and Pclogt/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 Pclowt/wt and Pclogt/gt neurons (left panel, Pclowt/wt = 0.27 ± 0.02, n = 27 primary dendrites; Pclogt/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.46629.004Figure 1—source data 1.This spreadsheet contains the normalized values used to generate the bar plots shown in Figure 1E,G and I.This spreadsheet contains the normalized values used to generate the bar plots shown in Figure 1E,G and I.Levels of synaptic vesicle proteins are reduced at Pclogt/gt synapses.(A) Immunocytochemical staining of cultured hippocampal neurons for SV proteins Synaptophysin, VGlut1, Synaptotagmin and Synapsin. The intensities of Synaptophysin, VGlut1 and Synaptotagmin are reduced in Pclogt/gt synapses. The levels of Synapsin, are not altered between Pclowt/wt and Pclogt/gt boutons. (B) Quantification of (A). (Synapotphysin : Pclowt/wt = 1.00 ± 0.01, n = 3457 puncta; Pclogt/gt = 0.81 ± 0.01, n = 2916 puncta; 13 independent experiments; VGlut1 : Pclowt/wt = 1.00 ± 0.02, n = 1907 puncta; Pclogt/gt = 0.70 ± 0.01, n = 2098 puncta; five independent experiments; Synaptotagmin : Pclowt/wt = 1.00 ± 0.01, n = 1987 puncta; Pclogt/gt = 0.69 ± 0.01, n = 1734 puncta; three independent experiments; Synapsin : Pclowt/wt = 1.00 ± 0.01, n = 2773 puncta; Pclogt/gt = 0.99 ± 0.01, n = 1848 puncta; four 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<0.0001.

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