Anti-Caveolin-1 抗体 - Caveolae Marker

• WB

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Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) at 1.5 µg/mL

Lane 1:

Human lung at 20 µg

Lane 2:

Human heart at 20 µg

Lane 3:

Human spleen at 20 µg

Secondary

All lanes:

Alexa Fluor anti-rabbit at 1/5000 dilution

Predicted band size: 20 kDa

Observed band size: 20 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

ab2910 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (ab255371) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab2910 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• ICC/IF

AbReview29088****

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

ab2910 staining Caveolin-1 - Caveolae Marker in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde. Samples were incubated with primary antibody (1/200 in PBS + 0.05% Saponin) for 1 hour at 37°C. A Cy3^®-conjugated Donkey anti-rabbit polyclonal (1/500) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Immunofluorescence analysis of Caveolin 1 was done on 70% confluent log phase A-375 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ab2910 at 1 μg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor^® 488 conjugate at a dilution of 1 : 2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor^® 555 Rhodamine Phalloidin. Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Rat astrocytes stained with fluorescently labeled Caveolin-1 antibody.

Primary antibody is ab2910 at a dilution of 1/500 and the secondary antibody is Texas red labeled anti-rabbit IgG at a dilution of 1/1000.

This image was kindly supplied as part of the review submitted by Donghui Zhu.

• IP

Supplier Data

Immunoprecipitation - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Caveolin 1 was immunoprecipitated using 5 μg of ab2910 from lysate of Mouse Heart (Lane 3) using the Dynabeads^® Protein A Immunoprecipitation Kit. Normal Rabbit IgG was used as a Isotype control (Lane 2). 10 % input represents the cell extract used for immunoprecipitation (Lane 1). Western blot analysis was performed using Caveolin 1 Rabbit Polyclonal Antibody and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

All lanes:

Immunoprecipitation - Anti-Caveolin-1 antibody - Caveolae Marker (ab2910)

Lane 1:

Input control

Lane 2:

Isotype control

Lane 3:

IP elute

Predicted band size: 20 kDa

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• WB

Supplier Data

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

The specificity of ab2910 was demonstrated by CRISPR targeted CAV1 knockout in HeLa cells. Western blot analysis of whole cell lysates using this antibody showed no detection of caveolin 1 protein expression in knockout cells compared to the protein detected at ~22kDa in wild-type HeLa cells.

Knockout validation info.

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) at 1 µg/mL

Lane 1:

CAV1 knockout HeLa cell lysate

Lane 2:

Wild-type HeLa cell lysate

Predicted band size: 20 kDa

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• WB

Supplier Data

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Western blot analysis was performed on whole cell extracts (20 μg lysate). The blots were probed with Anti-ab2910 (1-2 μg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. A 17 kDa band corresponding to Caveolin-1 was observed across cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex^® NuPAGE^® 12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex^® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot^® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) at 1 µg/mL

Lane 1:

PANC-1 (Human pancreatic epithelial cancinoma cell line) whole cell lysate at 20 µg

Lane 2:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 6:

U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg

Lane 7:

Mouse heart tissue lysate at 20 µg

Lane 8:

Rat heart tissue lysate at 20 µg

Lane 9:

C2C12 (Mouse myoblast cell line) whole cell lysate at 20 µg

Lane 10:

Mouse lung tissue lysate at 20 µg

Lane 11:

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

Predicted band size: 20 kDa

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• WB

Unknown

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

All lanes:

Western blot - Anti-Caveolin-1 antibody - Caveolae Marker (ab2910) at 2 µg/mL

All lanes:

Rat heart protein extract

Predicted band size: 20 kDa

false

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Immunocytochemistry-immunofluorescence using Anti-Caveolin-1 antibody - Caveolae Marker, ab2910. Publication image from Ruoslahti, E. et al., 2014, Nat Commun, 25277522. Legend direct from paper.

CendR endocytosis is mechanistically distinct from known endocytic pathways(A) The effect of knocking down selected hit genes from the genome screen on R-Ag and TFuptake using individual siRNAs format. The values represent relative probe uptakenormalized to that of negative controls (which is 1, Supplementary Data 2). The heat-map andclustering analysis were generated using Gene-E (Broad institute).(B) R-Ag uptake is not affected by Cav-ME and MP inhibitors. PPC1 cells were treated withindicated inhibitors (mβCD or Rottlerin) followed by testing for probe uptake(R-Ag, CtxB or Dex) as described in Methods. The fluorescence intensity of each probe wasnormalized to the average of the corresponding negative controls (vehicle alone) asrelative uptake (y-axis). *P<0.05 and **P<0.01(Student's t-test).(C) CendR cargo does not compete with other endocytic pathways. Unlabeled CendR peptide,RPARPAR-OH, was added to culture media of PPC1 cells at the indicated concentrations(upper right corner) 10 min prior to addition of fluorescently labeled endocytic probes(x-axis). The intensity of probe signal was normalized to the average of untreated cells(0 µM) as relative uptake (y-axis).(D) R-Ag does not colocalize with structural components of CME and Cav-ME vesicles. PPC1cells were incubated with R-Ag, TF-594 or CtxB (red) for 15 min before washing andfixation. CLTC and CAV1 proteins were detected with rabbit anti-CLTC and anti-CAV1antibodies, followed by staining with anti-rabbit secondary antibody (green). Nuclei werelabeled with Hoechst 33342 (blue). Representative images captured by confocal microscopyare shown. The colocalization events between two probes were identified with the“colocalization highlighter” macro for Image J and shown in yellow. Scalebar, 10 µm. Error bars indicate SEM (3-5 replicates).

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Immunocytochemistry-immunofluorescence using Anti-Caveolin-1 antibody - Caveolae Marker, ab2910. Publication image from Matthaeus, C. et al., 2022, Nat Commun, 36433988. Legend direct from paper.

Dynamin is not localized to caveolae.a Representative STED images of MEF membrane sheets expressing dynamin2-EGFP or dynamin2-K44A (in cyan), together with caveolin1 (magenta) and clathrin (yellow) immuno-staining. White arrows indicate dynamin localization. b Quantitative analysis of dynamin2 or dynamin2-K44A localization to caveolin1 spots in STED images illustrated in average STED fluorescence intensity projections (n(dynamin2) = 92, n(Dyn2-K44A) = 151, 3 independent experiments). c Percentage of caveolin1 spots that were also stained for cavin1, Dyn2-K44A or dynamin2. Bar plot indicates mean ± SE (n(cavin1) = 1135/8 cells, n(Dyn2-K44A) = 1283/11 cells, n(dynamin2) = 1032/8 cells, 3 independent experiments). d Representative STED-CLEM image showing Dyn2-K44A (cyan) and clathrin (yellow) on Pt replica TEM image. Increased image (I) illustrates caveolae, (II) shows clathrin vesicle (2 independent experiments). e Representative PREM image of membrane sheet obtained from dynamin triple knockout (dynamin1/2/3) MEFs. Zoom (III) illustrates increased membrane area covered with caveolae. f Total caveolae number at the plasma membrane in wild-type and dynamin triple knockout MEFs. Box plot shows mean ± SE, whiskers illustrate SD (n(wt) = 39 cell regions, n(Dyn 1/2/3 KO) = 13 cell regions, 2 independent experiments). g Percentage of caveolae types observed in plasma membrane sheets of wild-type and dynamin triple knockout MEFs. Bar graph indicates mean ± SE (n(wt) = 39 cell regions, n(Dyn 1/2/3 KO) = 13 cell regions, 2 independent experiments). h Radius of individual caveolae types (round caveolae domain was assumed), box plot shows mean ± SE, whiskers illustrate SD (caveolae number : wt : n(low) = 100, n(medium) = 113, n(high) = 96; Dyn 1/2/3 KO : n(low) = 50, n(medium) = 110, n(high) = 121, 2 independent experiments, significant difference was tested by two-sided Mann–Whitney test).

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Immunocytochemistry-immunofluorescence using Anti-Caveolin-1 antibody - Caveolae Marker, ab2910. Publication image from Matthaeus, C. et al., 2022, Nat Commun, 36433988. Legend direct from paper.

STED-CLEM revealed the localization of cavin proteins to low, medium and highly curved caveolae.a–b Representative CLEM images for MEFs expressing either caveolin1-EGFP (a) or caveolin2-EGFP (b) which were labelled with GFP nanobody-Atto647N and imaged with STED and PREM. c Quantitative analysis of low and highly curved caveolae STED fluorescence profiles from the center of caveolae to their edges (indicated by green dashed line, line graphs show mean ± SE; caveolae number : caveolin1 : n(low degree of invagination) = 145, n(high degree of invagination) = 178; caveolin2 : n(low) = 140, n(high) = 474, 4 independent experiments). d–f Representative CLEM images for MEFs expressing either cavin1-EGFP (d), cavin2-EGFP (e), or cavin3-EGFP (f) labelled with GFP nanobody-Atto647N and investigated by STED followed by PREM. g Quantitative analysis of low and highly curved caveolae by STED fluorescence profiles from the center of caveolae to their edges (indicated by green dashed line; line graphs show mean ± SE; caveolae number : cavin1 : n(low) = 59, n(high) = 272; cavin2 : n(low) = 231, n(high) = 223; cavin3 : n(low) = 198, n(high) = 191, 4 independent experiments). h Quantitative analysis of cavin1-3 localization to different caveolae types. Bar graph indicates caveolae stained positive for cavin1, 2 or 3 related to all caveolae per curvature type detected in CLEM images. Bar graph indicates mean ± SE; caveolae number : cavin1 : n = 821/9 cell regions; cavin2 : n = 1064/10 cell regions; cavin3 : n = 480/9 cell regions, 4 independent experiments).

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Caveolin-1 antibody - Caveolae Marker (AB2910)

Immunocytochemistry-immunofluorescence using Anti-Caveolin-1 antibody - Caveolae Marker, ab2910. Publication image from Matthaeus, C. et al., 2022, Nat Commun, 36433988. Legend direct from paper.

Stimulated emission depletion microscopy (STED) shows specific protein profiles for caveolae.a Confocal image of MEF plasma membrane sheet immunolabelled with an antibody against caveolin1 and a secondary anti-rabbit antibody tagged with Atto647N (Rb-Atto647N, 3 independent experiments). b Enlarged selection from (a) shows confocal and STED image of endogenous caveolin1 in MEFs. c Confocal and STED image of cavin1-EGFP expressing MEFs immunolabelled with caveolin1 antibody (secondary antibody dye Alexa594, magenta). Cavin1 was tagged by GFP nanobody labelled with Atto647N (blue, 3 independent experiments). d Zoomed STED image section from (c). STED fluorescence profile illustrates cavin1 localization to a caveolin1 spot. e Representative STED images of caveolae proteins (cyan) and caveolin1 antibody labeling (magenta, secondary antibody tagged with Alexa594) in plasma membrane sheets from MEFs. The individual caveolae proteins were expressed with EGFP tags and labelled with GFP nanobody-Atto647N. f Normalized average STED fluorescence intensity projection of automatically detected caveolin1 spots (magenta) and the corresponding co-labeled caveolae proteins (cyan). Lower panel shows both channels as merged image and the total number of caveolin1 spots is indicated. Scale bar represents 120 nm. g Percentage of caveolin1 spots that showed localization of either cavin1, EHBP1, EHD2 or pacsin2. Bar plot indicates mean ± SE (n(cavin1) = 1135/8 cells, n(EHBP1) = 1452/11 cells, n(EHD2) = 949/8 cells, n(pacsin2) = 1037/9 cells, 3 independent experiments). h The STED fluorescence plot profile for the individual caveolae proteins was analyzed from the center of the caveolin1 spot to the edge. Pixel size in STED images was 18.94 nm, based on the estimated caveolae diameter of 100 nm (radius = 50 nm), the edge of caveolae can be assumed between 3 and 4 pixel from the center (56–75 nm). i Fluorescence plot profiles from the center of caveolin1 spots accordingly to (h). Line graph indicates mean ± SE for each pixel and caveolae protein (n(caveolin1) = 121, n(caveolin2) = 71, n(cavin1) = 121, n(cavin2) = 85, n(cavin3) = 137, n(EHD2) = 136, n(pacsin2) = 94, n(EHBP1) = 127, 3 independent experiments).