Anti-VPS35 antibody [2D3]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VPS35 antibody [2D3] (AB57632)

ab57632 was shown to react with VPS35 in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a VPS35 knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab57632 at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

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.

• Flow Cyt

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Flow Cytometry - Anti-VPS35 antibody [2D3] (AB57632)

Overlay histogram showing HEK293 cells stained with ab57632 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab57632, 0.5μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This image was generated using a version of the antibody produced in ascites.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VPS35 antibody [2D3] (AB57632)

ab57632 staining VPS35 in wild-type HAP1 cells (top panel) and VPS35 knockout HAP1 cells (bottom panel). The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab57632 at 0.2 µg/mL and ab6046 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) (shown in pseudo colour green) and goat secondary antibody to Rabbit IgG (Alexa Fluor® 594) (ab150084) (shown in pseudo colour red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in pseudo colour blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-VPS35 antibody [2D3] (AB57632)

ab57632 was shown to react with VPS35 in wild-type HAP1 cells in Western blot with loss of signal observed in a VPS35 knockout cell line. Wild-type HAP1 and VPS35 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab57632 overnight at 4 °C at a 1/270 dilution. Blots were incubated with secondary antibodies at 0.2 µg/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-VPS35 antibody [2D3] (ab57632) at 1/270 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

VPS35 knock-out HAP1 lysate at 20 µg

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• WB

Lab

Western blot - Anti-VPS35 antibody [2D3] (AB57632)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : VPS35 knockout HAP1 cell lysate (20 μg)
Lane 3 : NIH/3T3 cell lysate (20 μg)
Lane 4 : A549 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab57632 observed at 91 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab57632 was shown to specifically react with VPS35 in wild-type HAP1 cells. No band was observed when VPS35 knockout samples were examined. Wild-type and VPS35 knockout samples were subjected to SDS-PAGE. ab5732 and ab181602 (loading control to GAPDH) were diluted 1μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

This image was generated using a version of the antibody produced in ascites.

All lanes:

Western blot - Anti-VPS35 antibody [2D3] (ab57632)

Predicted band size: 92 kDa

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• WB

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Western blot - Anti-VPS35 antibody [2D3] (AB57632)

Western blot against tagged recombinant protein immunogen using ab57632 VPS35 antibody at 1 μg/ml. Predicted band size of immunogen is 37 kDa

This image was generated using a version of the antibody produced in ascites.

All lanes:

Western blot - Anti-VPS35 antibody [2D3] (ab57632)

Predicted band size: 92 kDa

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• WB

CiteAb

Western blot - Anti-VPS35 antibody [2D3] (AB57632)

Western Blotting using Anti-VPS35 antibody [2D3], ab57632. Publication image from Seaman, M. N. et al., 2014, Nat Commun, 24819384. Legend direct from paper.

VPS35 D620N impairs autophagy, while VPS35 knockdown has only modest effects.(a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 x 10−6 (DMSO) and 3.81 x 10−5 (Baf) by 2-tailed Student’s t-test. (c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P<0.0001 by 2-tailed Student’s t-test. (d) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student’s t-test. (e) Confocal images representative of the experiment described in d. Scale bar, 20 µm. (f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting. (g) Quantification of the representative experiment in triplicate in f, in which the level ofα-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student’s t-test. (h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate. (i) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student’s t-test. (j) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.

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• WB

CiteAb

Western blot - Anti-VPS35 antibody [2D3] (AB57632)

Western Blotting using Anti-VPS35 antibody [2D3], ab57632. Publication image from Seaman, M. N. et al., 2014, Nat Commun, 24819384. Legend direct from paper.

VPS35 D620N impairs autophagy, while VPS35 knockdown has only modest effects.(a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 x 10−6 (DMSO) and 3.81 x 10−5 (Baf) by 2-tailed Student’s t-test. (c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P<0.0001 by 2-tailed Student’s t-test. (d) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student’s t-test. (e) Confocal images representative of the experiment described in d. Scale bar, 20 µm. (f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting. (g) Quantification of the representative experiment in triplicate in f, in which the level ofα-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student’s t-test. (h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate. (i) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student’s t-test. (j) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.

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