Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain

doi: 10.1177/0271678x16661728

Figure Lengend Snippet: Figure 5. SDPR traffics to the lumen of microvessel endothelial cells after a peripheral inflammatory pain stimulus. (a) Representative fluorescence microscopy images of a microvessel section stained for SDPR (green) and GLUT1 (red) with a DAPI (blue: nuclear) counterstain compared with the no primary control. Images were acquired using a 40 objective. Bars represent 20 mm. (b) Representative deconvolution microscopy images stained for SDPR (green) with a DAPI (blue: nuclear) counterstain. Image was acquired using a 60 objective. Bars represent 5 mm. Abbreviations: pericytes (P); lumen (L). (c) Representative deconvolution microscopy images of microvessels in saline (SAL)-injected (control) and carrageenan (CAR)-injected animals stained for SDPR (green) and GLUT1 (red) with a DAPI (blue: nuclear) counterstain. Grayscale images are SDPR only. Images were acquired using a 60 objective. Bars represent 5 mm. (d) Colocalization analysis of the overlap of the SDPR signal with the GLUT1 and DAPI signals in microvessel endothelial cells. Values are the mean þ SEM (n ¼ 30 (GLUT1) or 32 (DAPI) total images representing vessels from nine animals/treatment). * indicates significantly different from SAL values for GLUT1 (p ¼ 2.37E-09) or DAPI (p ¼ 1.77E-02). (e) Representative fluorescence microscopy image of a microvessel section stained for PgP (ab3366: green) and SDPR (red) with a DAPI (blue: nuclear) counterstain compared with the no primary control. Images were acquired using a 40 objective. Bars represent 20 mm. (f) Representative deconvolution microscopy images of microvessels in SAL-injected and CAR-injected animals stained for PgP (green) and SDPR (red) with a DAPI (blue: nuclear) counterstain. Images were acquired using a 60 objective. Bars represent 5 mm.

Article Snippet: The following primary antibodies and AlexaFluorlabeled secondary antibodies were used for staining: MDR1 (orb11267; 1:5 dilution) from Biorbyt (San Francisco, CA); MDR1 (ab3366; 1:5 dilution), GLUT1 (ab32551; 1:10 dilution), GLUT1 (ab40084; 1:10 dilution), PTRF/cavin1 (ab48824; 1:5 dilution), and von Willebrand factor (vWF; ab6994; 1:10 dilution) from AbCam (Cambridge, MA); CAV1 (BD606100; 1:20 dilution) from BD Biosciences (San Jose, CA); SDPR/cavin2 (SDPR-12339-1-AP; 1:5 dilution) and protein kinase C delta binding protein (PRKCDBP)/cavin3 (PRKCDBP-16250-1-AP; 1:10 dilution) from ProteinTech (ThermoFisher, Waltham, MA); and AlexaFluor488 goat anti-rabbit (A-11008; 1:20 dilution), AlexaFLuor488 goat anti-mouse (A-11001; 1:20 dilution), AlexaFLuor568 goat antirabbit (A-11011; 1:20 dilution) and AlexaFLuor568 goat anti-mouse (A-11004; 1:20 dilution) from Life Technologies (Carlsbad, CA).

Techniques: Fluorescence, Microscopy, Staining, Control, Saline, Injection

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain

doi: 10.1177/0271678x16661728

Figure Lengend Snippet: Figure 7. Model indicating PgP trafficking from the nucleus to the luminal membrane of the microvessel endothelial cells after a peripheral inflammatory pain (PIP) stimulus. In control animals, PgP is located in the nuclear membrane and the plasma membrane in caveolar and non-caveolar regions. After a PIP stimulus, PgP decreases in the nuclear membrane suggesting that PgP traffics from the nuclear membrane to the plasma membrane. PIP: peripheral inflammatory pain; PgP: P-glycoprotein; PTRF/cavin1: polymerase 1 and transcript release factor; SDPR/cavin2: serum deprivation response protein.

Article Snippet: The following primary antibodies and AlexaFluorlabeled secondary antibodies were used for staining: MDR1 (orb11267; 1:5 dilution) from Biorbyt (San Francisco, CA); MDR1 (ab3366; 1:5 dilution), GLUT1 (ab32551; 1:10 dilution), GLUT1 (ab40084; 1:10 dilution), PTRF/cavin1 (ab48824; 1:5 dilution), and von Willebrand factor (vWF; ab6994; 1:10 dilution) from AbCam (Cambridge, MA); CAV1 (BD606100; 1:20 dilution) from BD Biosciences (San Jose, CA); SDPR/cavin2 (SDPR-12339-1-AP; 1:5 dilution) and protein kinase C delta binding protein (PRKCDBP)/cavin3 (PRKCDBP-16250-1-AP; 1:10 dilution) from ProteinTech (ThermoFisher, Waltham, MA); and AlexaFluor488 goat anti-rabbit (A-11008; 1:20 dilution), AlexaFLuor488 goat anti-mouse (A-11001; 1:20 dilution), AlexaFLuor568 goat antirabbit (A-11011; 1:20 dilution) and AlexaFLuor568 goat anti-mouse (A-11004; 1:20 dilution) from Life Technologies (Carlsbad, CA).

Techniques: Membrane, Control, Clinical Proteomics

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) HeLa cells were cross-linked with DSP and solubilised in 1% Triton X-100/1% octyl glucoside. Lysates were fractionated on 10–40% sucrose gradients, followed by Western blotting of gradient fractions 1–12 using antibodies against caveolin 1, cavin 1, GFP, clathrin heavy chain, or flotillin 2. Gradients were prepared from cells expressing caveolin-1-GFP (top panel) or flotillin-2-GFP as a control (middle panel). The bottom panel shows a gradient from flotillin-2-GFP expressing cells that had not been cross-linked prior to cell lysis. The high molecular weight (HMW) peak 8–10 of caveolin 1 and cavin 1 is boxed. The distribution of molecular weight protein standards in the gradients is indicated on the bottom. (B) Caveolin-1-GFP or flotillin-2-GFP (to provide a control) was immuno-isolated from pooled gradient fractions 8–10, indicated “HMW” in (A). A control immuno-isolation was also performed from fractions 8–10 of cells expressing GFP. Eluted proteins were visualized by silver staining. Cavin 1, caveolin-1-GFP, cavin 3, and caveolins are indicated. The bands specific to the flotillin-2-GFP immunoprecipitation, marked with asterisks (*) are flotillin-2-GFP, and endogenous flotillins 1 and 2. (C) The caveolin 1 HMW complex was analyzed by LC-MS/MS, and the graph shows the number of peptides for each protein identified. Proteins are ranked left to right by number of peptides identified. (D) Western blots of the starting material (In) and unbound material (Un) after immuno-isolation of caveolin-1-GFP, flotillin-2-GFP, or GFP from pooled HMW fractions 8–10. Membranes were probed with anti-GFP or anti-cavin 1 antibodies.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Western Blot, Expressing, Lysis, Molecular Weight, Isolation, Silver Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) Cross-linked and detergent solubilised (1% Triton X-100/1% octyl glucoside) HeLa cell extracts were fractionated by velocity centrifugation (10–40% sucrose), followed by Western blotting of fractions 1–12. Gradients were prepared from cells expressing either GFP (the control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, cavin 3, or GFP. The high molecular weight (HMW) peak of caveolin 1 and cavins is boxed. The strong band in the cavin 3 blot (marked with an asterisk (*); fractions 1–4) are nonspecific: they are also observed in cavin 3 knockout cells and in HeLa cells depleted of cavin 3 by siRNA (see ). (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin 1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. Control was GFP-expressing cells. (C) Cavin 1, 2, and-3-GFP were immuno-isolated from pooled gradient HMW fractions 8–10. Control immuno-isolations were performed from pooled fractions 8–10 of cells expressing GFP or flotillin-2-GFP. Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, caveolin 1, EHD2, and pacsin 2. Note the absence of EHD2 and pacsin 2 from the caveolar coat complex. (D) HMW complexes immuno-isolated from cross-linked HeLa cells expressing caveolin-1-GFP, or cavin 1, 2, or-3-GFP were analyzed by LC-MS/MS. The graph is showing the number of peptides identified from caveolin 1 and cavin proteins in each complex. Other proteins identified by mass spectrometry are not shown.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Centrifugation, Western Blot, Expressing, Molecular Weight, Knock-Out, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) Gradient fractions (10–40% sucrose) of non-cross-linked and detergent solubilised (1% Triton X-100) HeLa cell extracts were analysed by Western blotting. Gradients were prepared from cells expressing either GFP (as control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, or GFP. Pooled fractions 3–5 and 6–8 used for immuno-isolation shown in (C) are boxed. (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin-1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. (C) Cavin 1, 2, and -3-GFP, flotillin-2-GFP, caveolin-1-GFP, or GFP were immuno-isolated from gradient fractions 3–5 or 6–8 as shown in (A). Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, or caveolin 1. (D) Caveolar coat complexes immuno-isolated from cross-linked HeLa cells stably expressing cavin-2-GFP were incubated with increasing concentrations of DTT to partially reduce crosslinks and analyzed by Western blotting using anti-caveolin 1 or anti-cavin 1 antibodies. Monomeric and oligomeric species of caveolin 1 and cavin 1 are indicated. Note the 180 kDa cavin 1 trimer.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Western Blot, Expressing, Isolation, Stable Transfection, Incubation

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) Western blotting of cell lysates from siRNA-treated cells. Blots were probed with the anitbodies indicated. * indicates the nonspecific band obtained with anti-cavin-3 antibodies. (B) Western blotting of fractions 3 to 5 (LMW pool) and 8 to 10 (HMW pool: these fractions contain the caveolar coat complex) of sucrose velocity gradients analyzing the distribution of cavins and caveolin 1 from cells treated with the siRNAs shown. Blots of the entire gradients are shown in . (C) Quantification of the ratio between the amount of cavin 1 and caveolin 1 in fractions and 8 to 10 (HMW pool: these fractions contain the caveolar coat complex) of sucrose velocity gradients as shown in the blots in (B). Quantification is from densitometric scans of Western blots, and is based on three separate experiments; bars are SEM. In each experiment, all values were normalized so that the intensity of relevant bands in control flotillin 1 and 2 siRNA treated samples was 1.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Western Blot

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) HeLa cell lines stably transfected with either caveolin-1-GFP, cavin 1, 2, or-3-GFP were processed for immuno-electron microscopy, using anti-GFP primary antibodies, nanogold-conjugated secondary antibodies, and silver enhancement. Representative transmission EM images showing specific immuno-labeling of caveolin-1-GFP. (B) Membrane profiles and the localisation of gold particles (as shown in A) from about 50 caveolae per cell line as shown were superimposed; labeling was with anti-GFP antibodies as in (A). (C) HeLa cells were processed for immuno-electron microscopy using anti-caveolin 1 or cavin 1 primary antibodies, nanogold-conjugated secondary antibodies, and silver enhancement. Images represent superimposition of around 50 caveolae. (D) Membrane profiles and the localisation of gold particles (as shown in A) from about 50 caveolae from GFP-EHD2 expressing cells were superimposed; labeling was with anti-GFP antibodies as in (A).

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Stable Transfection, Transfection, Immuno-Electron Microscopy, Transmission Assay, Immunolabeling, Labeling, Expressing

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) HeLa cells were cross-linked with DSP and solubilised in 1% Triton X-100/1% octyl glucoside. Lysates were fractionated on 10–40% sucrose gradients, followed by Western blotting of gradient fractions 1–12 using antibodies against caveolin 1, cavin 1, GFP, clathrin heavy chain, or flotillin 2. Gradients were prepared from cells expressing caveolin-1-GFP (top panel) or flotillin-2-GFP as a control (middle panel). The bottom panel shows a gradient from flotillin-2-GFP expressing cells that had not been cross-linked prior to cell lysis. The high molecular weight (HMW) peak 8–10 of caveolin 1 and cavin 1 is boxed. The distribution of molecular weight protein standards in the gradients is indicated on the bottom. (B) Caveolin-1-GFP or flotillin-2-GFP (to provide a control) was immuno-isolated from pooled gradient fractions 8–10, indicated “HMW” in (A). A control immuno-isolation was also performed from fractions 8–10 of cells expressing GFP. Eluted proteins were visualized by silver staining. Cavin 1, caveolin-1-GFP, cavin 3, and caveolins are indicated. The bands specific to the flotillin-2-GFP immunoprecipitation, marked with asterisks (*) are flotillin-2-GFP, and endogenous flotillins 1 and 2. (C) The caveolin 1 HMW complex was analyzed by LC-MS/MS, and the graph shows the number of peptides for each protein identified. Proteins are ranked left to right by number of peptides identified. (D) Western blots of the starting material (In) and unbound material (Un) after immuno-isolation of caveolin-1-GFP, flotillin-2-GFP, or GFP from pooled HMW fractions 8–10. Membranes were probed with anti-GFP or anti-cavin 1 antibodies.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Western Blot, Expressing, Lysis, Molecular Weight, Isolation, Silver Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) Cross-linked and detergent solubilised (1% Triton X-100/1% octyl glucoside) HeLa cell extracts were fractionated by velocity centrifugation (10–40% sucrose), followed by Western blotting of fractions 1–12. Gradients were prepared from cells expressing either GFP (the control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, cavin 3, or GFP. The high molecular weight (HMW) peak of caveolin 1 and cavins is boxed. The strong band in the cavin 3 blot (marked with an asterisk (*); fractions 1–4) are nonspecific: they are also observed in cavin 3 knockout cells and in HeLa cells depleted of cavin 3 by siRNA (see ). (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin 1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. Control was GFP-expressing cells. (C) Cavin 1, 2, and-3-GFP were immuno-isolated from pooled gradient HMW fractions 8–10. Control immuno-isolations were performed from pooled fractions 8–10 of cells expressing GFP or flotillin-2-GFP. Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, caveolin 1, EHD2, and pacsin 2. Note the absence of EHD2 and pacsin 2 from the caveolar coat complex. (D) HMW complexes immuno-isolated from cross-linked HeLa cells expressing caveolin-1-GFP, or cavin 1, 2, or-3-GFP were analyzed by LC-MS/MS. The graph is showing the number of peptides identified from caveolin 1 and cavin proteins in each complex. Other proteins identified by mass spectrometry are not shown.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Centrifugation, Western Blot, Expressing, Molecular Weight, Knock-Out, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) Gradient fractions (10–40% sucrose) of non-cross-linked and detergent solubilised (1% Triton X-100) HeLa cell extracts were analysed by Western blotting. Gradients were prepared from cells expressing either GFP (as control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, or GFP. Pooled fractions 3–5 and 6–8 used for immuno-isolation shown in (C) are boxed. (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin-1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. (C) Cavin 1, 2, and -3-GFP, flotillin-2-GFP, caveolin-1-GFP, or GFP were immuno-isolated from gradient fractions 3–5 or 6–8 as shown in (A). Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, or caveolin 1. (D) Caveolar coat complexes immuno-isolated from cross-linked HeLa cells stably expressing cavin-2-GFP were incubated with increasing concentrations of DTT to partially reduce crosslinks and analyzed by Western blotting using anti-caveolin 1 or anti-cavin 1 antibodies. Monomeric and oligomeric species of caveolin 1 and cavin 1 are indicated. Note the 180 kDa cavin 1 trimer.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Western Blot, Expressing, Isolation, Stable Transfection, Incubation

Journal: PLoS Biology

Article Title: Molecular Composition and Ultrastructure of the Caveolar Coat Complex

doi: 10.1371/journal.pbio.1001640

Figure Lengend Snippet: (A) Western blotting of cell lysates from siRNA-treated cells. Blots were probed with the anitbodies indicated. * indicates the nonspecific band obtained with anti-cavin-3 antibodies. (B) Western blotting of fractions 3 to 5 (LMW pool) and 8 to 10 (HMW pool: these fractions contain the caveolar coat complex) of sucrose velocity gradients analyzing the distribution of cavins and caveolin 1 from cells treated with the siRNAs shown. Blots of the entire gradients are shown in . (C) Quantification of the ratio between the amount of cavin 1 and caveolin 1 in fractions and 8 to 10 (HMW pool: these fractions contain the caveolar coat complex) of sucrose velocity gradients as shown in the blots in (B). Quantification is from densitometric scans of Western blots, and is based on three separate experiments; bars are SEM. In each experiment, all values were normalized so that the intensity of relevant bands in control flotillin 1 and 2 siRNA treated samples was 1.

Article Snippet: The following antibodies were used: Mouse anti-GFP (Roche, 11814460001), rabbit anti-PTRF (cavin 1) (Abcam, ab48824), rabbit anti-SRBC (PRKCDBP; cavin 3) (Abcam, ab83913), goat anti-SDPR (cavin 2) (R&D Systems, AF5759), goat anti-EHD2 (Abcam, ab23935), rabbit anti-Caveolin 1 (BD, 610060), mouse anti-flotillin-1 (BD, 610821), mouse anti-flotillin-2 (BD, 610384), mouse anti-clathrin heavy chain (×11), rabbit anti-GFP antibody (Abcam, ab6556), and rabbit anti-RFP (MBL, PM005).

Techniques: Western Blot

Journal: eLife

Article Title: Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination

doi: 10.7554/eLife.29854

Figure Lengend Snippet:

Article Snippet: Polyclonal rabbit anti-CAVIN-1 and 3 antibodies (18892–1-AP; 16250–1-AP) were from Proteintech, polyclonal goat anti-CAVIN-2 antibodies (AF5759) were from R&D Systems and polyclonal rabbit anti-CAVIN-4 antibodies (HPA020987) were from Sigma.

Techniques: Transfection, Construct, Western Blot, Recombinant, Plasmid Preparation, Software, Staining