重组Anti-VPS26抗体[EPR13456]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-VPS26 antibody [EPR13456] (AB181352)

Immunofluorescent analysis of HeLa cells labeling VPS26 with ab181352 at 1/100 dilution (red). DAPI nuclear staining (blue).

• IP

Supplier Data

Immunoprecipitation - Anti-VPS26 antibody [EPR13456] (AB181352)

Western blot analysis on immunoprecipitation pellet from HeLa cell lysate pulling down VPS26 using ab181352 at 1/10 dilution.

All lanes:

Immunoprecipitation - Anti-VPS26 antibody [EPR13456] (ab181352)

Predicted band size: 38 kDa

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• WB

Supplier Data

Western blot - Anti-VPS26 antibody [EPR13456] (AB181352)

All lanes:

Western blot - Anti-VPS26 antibody [EPR13456] (ab181352) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

293T cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Predicted band size: 38 kDa

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• WB

Lab

Western blot - Anti-VPS26 antibody [EPR13456] (AB181352)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : VPS26 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : HepG2 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab181352 observed at 40 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab181352 was shown to specifically react with VPS26 when VPS26 knockout samples were used. Wild-type and VPS26 knockout samples were subjected to SDS-PAGE. ab181352 and ab18058 (loading control to Vinculin) were diluted to 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ( ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-VPS26 antibody [EPR13456] (ab181352)

Predicted band size: 38 kDa

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• WB

CiteAb

Western blot - Anti-VPS26 antibody [EPR13456] (AB181352)

Western Blotting using Anti-VPS26 antibody [EPR13456], ab181352. Publication image from Jimenez-Orgaz, A. et al., 2018, EMBO J, 29158324. Legend direct from paper.

Control of RAB7 activity is not required for retromer‐based sorting of integral membrane proteinsAll images show formaldehyde‐fixed cells. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were co‐stained for endogenous GLUT1 (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments.Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were surface‐biotinylated, followed by streptavidin isolation and Western blot‐based quantification of biotinylated surface proteins. Surface GLUT1 was quantified over four independent experiments.RAB7a knockout cells and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs cells were co‐stained for endogenous GLUT1 (red) and endogenous LAMP2 (blue), and co‐localization was quantified over two independent experiments.Parental HeLa cells, RAB7a knockout cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were co‐stained for endogenous CI‐MPR (red) and endogenous TGN46 (blue).Data information : All scale bars = 10 µm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells. Source data are available online for this figure.

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• WB

CiteAb

Western blot - Anti-VPS26 antibody [EPR13456] (AB181352)

Western Blotting using Anti-VPS26 antibody [EPR13456], ab181352. Publication image from Jimenez-Orgaz, A. et al., 2018, EMBO J, 29158324. Legend direct from paper.

TBC1D5 and retromer cooperate in the control of RAB7 activity and mobilityGFP‐trap IPs of the indicated GFP‐tagged VPS29 (upper panel) or TBC1D5 (lower panel) constructs confirm that the VPS29‐L152E and the TBC1D5‐L142E mutant lose binding to each other.Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Note that re‐expression of both VPS29 variants fully restores the level of endogenous VPS35.PFA‐fixed parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments.Parental HeLa cells and VPS29 KO cells as well as VPS29 KO cells transduced with the indicated VPS29 rescue constructs were transduced with GFP‐RAB7, and RAB7 mobility/turnover was analyzed by FRAP in living cells. The displayed recovery kinetics were obtained by averaging kinetics from fifteen FRAP recoveries per condition acquired in two independent experiments.Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments.Parental HeLa cells and clonal TBC1D5 KO cells and TBC1D5 KO cells transduced with the indicated GFP‐TBC1D5 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay.Data information : All scale bars = 10 µm, all error bars = SD, and *P < 0.05 in a t‐test of the respective condition compared to the control cells. Source data are available online for this figure.

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