Anti-Caveolin-1 antibody - Caveolae Marker

• IP

Unknown

Immunoprecipitation - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

Caveolin 1 - Caveolae Marker was immunoprecipitated using 0.5mg Mouse Heart whole tissue lysate, 5µg of Rabbit polyclonal to Caveolin 1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Heart whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18199.
Secondary : Clean blot (HRP conjugate) at 1/1000 dilution.
Band : 20kDa : Caveolin 1 - Caveolae Marker.

All lanes:

Immunoprecipitation - Anti-Caveolin-1 antibody - Caveolae Marker (ab18199)

Predicted band size: 20 kDa

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• WB

Lab

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

Lanes 1 - 4 : Merged signal (red and green). Green - ab18199 observed at 21-24 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab18199 was shown to react with Caveolin-1 in wild-type A431 cells in western blot. Loss of signal was observed when CAV1 knockout sample was used. Wild-type and CAV1 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab18199 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/mL

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

CAV1 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CAV1 (Caveolin-1) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-cav1-caveolin-1-knockout-a-431-cell-line-ab269583'>ab269583</a>)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 20 kDa

Observed band size: 21-24 kDa

false

• WB

Project

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/mL

Lane 1:

Western blot - NIH/3T3 whole cell lysate (<a href='/en-us/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 10 µg

Lane 2:

MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 3:

Heart (Mouse) Tissue Lysate at 10 µg

Lane 4:

Heart (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 20 kDa

Observed band size: 20 kDa

false

• WB

Project

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/mL

Lane 1:

Mouse Heart Lysate at 10 µg

Lane 2:

Human Heart Lysate at 10 µg

Secondary

All lanes:

Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/5000 dilution

Predicted band size: 20 kDa

Observed band size: 20 kDa

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• WB

Unknown

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

ab18199 recognizes the full length tagged recombinant Caveolin 1 protein (ab114170) which has an expected molecular weight of 46 kDa.

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/mL

All lanes:

Western blot - Recombinant Human Caveolin-1 protein (GST tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-caveolin-1-protein-ab114170'>ab114170</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 20 kDa

true

Exposure time: 1min

• WB

Unknown

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

ab18199 recognizes the full length tagged recombinant Caveolin 1 protein (ab114170) which has an expected molecular weight of 46 kDa.

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab18199) at 1 µg/mL

All lanes:

Western blot - Recombinant Human Caveolin-1 protein (GST tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-caveolin-1-protein-ab114170'>ab114170</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 20 kDa

true

Exposure time: 1min

• WB

CiteAb

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

Western Blotting using Anti-Caveolin-1 antibody - Caveolae Marker, ab18199. Publication image from Takahashi, T. et al., 2016, Nat Commun, 26725982. Legend direct from paper.

ROR1 sustains CAV1 expression through cavin-1 and CAV1 binding.(a) Indispensable involvement of the cavin-1-binding region of ROR1 in sustaining CAV1 expression shown in PC-9 cells reconstituted with ROR1 lacking the cavin-1-binding region. (b) Indispensable involvement of the CAV1 binding region of ROR1 for sustained CAV1 expression shown in PC-9 cells reconstituted with ROR1 lacking the CAV1-binding region. Uncropped images of blots are shown in Supplementary Fig. 11.

false

• WB

CiteAb

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

Western Blotting using Anti-Caveolin-1 antibody - Caveolae Marker, ab18199. Publication image from Takahashi, T. et al., 2016, Nat Commun, 26725982. Legend direct from paper.

ROR1 kinase activity is not required to sustain CAV1 expression.(a) Sustainment of CAV1 expression in the presence of siROR1 by introduction of both siRNA-resistant, wild-type (wt) and kinase-dead ROR1 in the NCI-H1975 cells. Also see Supplementary Fig. 7. (b) Sucrose density-gradient assay showing that ROR1 kinase activity is dispensable for the retention of cavin-1 in DRM fractions. NCI-H1975 cells stably transfected with either wt or kinase-dead forms of siRNA-resistant ROR1 were subjected to siROR1 treatment in this analysis. Uncropped images of blots are shown in Supplementary Fig. 11.

false

• WB

CiteAb

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

Western Blotting using Anti-Caveolin-1 antibody - Caveolae Marker, ab18199. Publication image from Takahashi, T. et al., 2016, Nat Commun, 26725982. Legend direct from paper.

ROR1 inhibits the lysosomal degradation of CAV1 and sustains intact caveolae structures.(a) Decreased CAV1 but not of CAV2 expression with the use of three independent ROR1 siRNAs. See Supplementary Figs 2a–e and 3, which shows similar effects in other cell lines. (b) Reduced CAV1 expression by knocking down ROR1 but not EGFR, ERBB2 or MET in NCI-H1975 cells. Note that cavin-1 expression is not affected. (c) Rescue of CAV1 expression by treatment with a lysosome inhibitor (left panel) but not by treatment with a proteasome inhibitor (right panel) in NCI-H1975 cells knocked down for ROR1. (d) Schematic diagram of immunoelectron microscopy of SDS-treated freeze-fracture replica. (e) Decreased number of typical caveolae structures in the plasma membranes of the siROR1-treated NCI-H1975 cells were observed in the electron microscopic examination. More than 18 random plasma membrane areas of 17–50 µm2 were examined for both samples. (average±s.e.m.; Student’s t-test; *P<0.001.) (f) Representative results of the freeze-fracture immunoelectron microscopy. CAV2 labels made clusters, but the labelled membrane was either flat or superficially depressed in the siROR1-treated NCI-H1975 cells. By contrast, the CAV2 labelling in the siControl-treated NCI-H1975 cells showed intact caveolae with indentations measuring 50–75 nm in diameter. Also refer to Supplementary Fig. 4. Uncropped images of blots are shown in Supplementary Fig. 11.

false

• WB

CiteAb

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB18199)

Western Blotting using Anti-Caveolin-1 antibody - Caveolae Marker, ab18199. Publication image from Takahashi, T. et al., 2016, Nat Commun, 26725982. Legend direct from paper.

ROR1 inhibits the lysosomal degradation of CAV1 and sustains intact caveolae structures.(a) Decreased CAV1 but not of CAV2 expression with the use of three independent ROR1 siRNAs. See Supplementary Figs 2a–e and 3, which shows similar effects in other cell lines. (b) Reduced CAV1 expression by knocking down ROR1 but not EGFR, ERBB2 or MET in NCI-H1975 cells. Note that cavin-1 expression is not affected. (c) Rescue of CAV1 expression by treatment with a lysosome inhibitor (left panel) but not by treatment with a proteasome inhibitor (right panel) in NCI-H1975 cells knocked down for ROR1. (d) Schematic diagram of immunoelectron microscopy of SDS-treated freeze-fracture replica. (e) Decreased number of typical caveolae structures in the plasma membranes of the siROR1-treated NCI-H1975 cells were observed in the electron microscopic examination. More than 18 random plasma membrane areas of 17–50 µm2 were examined for both samples. (average±s.e.m.; Student’s t-test; *P<0.001.) (f) Representative results of the freeze-fracture immunoelectron microscopy. CAV2 labels made clusters, but the labelled membrane was either flat or superficially depressed in the siROR1-treated NCI-H1975 cells. By contrast, the CAV2 labelling in the siControl-treated NCI-H1975 cells showed intact caveolae with indentations measuring 50–75 nm in diameter. Also refer to Supplementary Fig. 4. Uncropped images of blots are shown in Supplementary Fig. 11.

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