mouse monoclonal anti cav1 antibody  (Proteintech)

Journal: bioRxiv

Article Title: Galectin-1 Modulates Cell Adhesions, Caveolae, and Vascular Permeability in Kidney Endothelial Cells – Insights from Proteomics, Phosphoproteomics, and Functional Studies

doi: 10.64898/2026.03.03.709385

Figure Lengend Snippet: A) Overview of treatment and phosphoproteome enrichment workflow. B) Phosphopeptides identified, quantified, and significant for each treatment and their interaction after correcting for matched unmodified peptides using MSStatsPTM in R. C) Volcano plots of phosphopeptide Log2FC of IFNγ treatment, LGALS1 siRNA, and both IFNγ and LGALS1 siRNA, compared to non-targeting control and vehicle plotted against –log10(p-value). Orange colour represents non-significant proteins, while blue colour represents those with p<0.01 (linear mixed-effects model), and green represents those with FDR: BH<0.2. D) Heatmap of 28 phosphopeptides significant in at least 1 comparison from panel C. Red colour indicates increased expression, blue colour indicates decreased expression, and black colour indicates phosphopeptides that were not found in that comparison. Colour on the top bar denotes function (obtained from uniport.org ) of the protein the phosphopeptide belongs to. Black and grey asterisks denote FDR<0.2 and p<0.01, respectively. Immunostaining of CAVN1 and CAV1 when GMECs were treated with LGALS1 siRNA, IFNγ, or both. Green colour indicates CAV1, red colour indicates CAVN1. Red arrows denote co-staining of CAV1 and CAVN1 in the NT_V condition. LGALS1 , galectin-1 gene; GMEC, glomerular microvascular endothelial cells; IFNγ, interferon-γ; NT, non-targeting control; NT_V, non-targeting control and vehicle; PTM, post-translational modification.

Article Snippet: Samples were incubated overnight at 4°C with rabbit polyclonal anti-galectin-1 antibody (1:100; GTX101566; GeneTex), rabbit polyclonal anti-CAVN1 antibody (1:1000; 18892-1-AP; Proteintech), or mouse monoclonal anti-CAV1 antibody (1:1000; 66067-1-IG; Proteintech).

Techniques: Phospho-proteomics, Control, Comparison, Expressing, Immunostaining, Staining, Modification

Journal: Communications biology

Article Title: The Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling in pulmonary hypertension by interfering with BMPR2/Caveolin-1 binding.

doi: 10.1038/s42003-023-05693-2

Figure Lengend Snippet: Fig. 1 Caveolae and BMP/Smad signaling in CAV1−/−PAECs and PASMCs. a Representative electron micrographs of PAECs and PASMCs in lungs from WT mice and CAV1−/−mice. Number of caveolae in PAECs and PASMCs of WT mice and CAV1−/−mice were calculated. Black arrows indicate caveolae. Scale bar, 200 nm. *P < 0.05 compared with WT. b PMVECs were isolated from the lung of WT mice and CAV1−/−mice. Expression of caveolae- associated proteins and BMP/Smad signaling were assessed in WT and CAV1−/−PMVECs. *P < 0.05 compared with WT. ns, not significant. c PASMCs were isolated from the lung of WT and CAV1−/−mice. Expression of caveolae-associated proteins and BMP/Smad signaling were assessed in WT and CAV1−/−PASMCs. ns not significant. Data are expressed as the mean ± standard error.

Article Snippet: The rabbit polyclonal anti-CAV1 antibody (#sc-894), the mouse monoclonal anti-CAV1 antibody (#sc-70516), the monoclonal anti-BMPR1a antibody (#sc-518037) and the mouse monoclonal anti-VE cadherin antibody (#sc-9989) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); the rabbit polyclonal antibody to Cavin-1 (#ab76919) and αSMA (#ab124964), the horseradish peroxidase-conjugated monoclonal antibody to GAPDH (#ab105428) and the mouse monoclonal antibody to myc tag (#ab32) were from Abcam PLC (Cambridge, UK); the rabbit polyclonal antibodies to Akt (#9272), phosphoAkt (Ser473) (#9271), phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad9 (Ser465/467) (#13820), Smad1 (#9743), phospho-Smad2 (Ser465/467) (#3108), Smad2/3 (#3102) and the horseradish peroxidase-conjugated secondary antibodies (antimouse-HRP, and anti-rabbit-HRP) were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit polyclonal antibody to Cavin-2 (#12339-1-AP) and Cavin-3 (PRKCDBP) (#16250-1-AP) was purchased from ProteinTech Group, Inc. (Rosemont, IL, USA); the rabbit polyclonal anti-Cavin-4 antibody was generated as previously described58,59; the mouse monoclonal antibody to FLAG (clone M2, #F3165,), the mouse monoclonal antibody to T7 (#69522), Cy3-conjugated monoclonal antibody to αSMA (#C6198) and collagenase type 1 from Clostridium histolyticum were from Merck (Darmstadt, Germany); the mouse monoclonal antibodies to CAV3 (#610421) and the rat monoclonal antibodies to CD31 (clone MEC13.3, #550274) were from BD Biosciences (Franklin Lakes, NJ, USA); the horseradish peroxidase-conjugated monoclonal antibody to GST (#NB600-388) was from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Isolation, Expressing

Journal: Communications biology

Article Title: The Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling in pulmonary hypertension by interfering with BMPR2/Caveolin-1 binding.

doi: 10.1038/s42003-023-05693-2

Figure Lengend Snippet: Fig. 2 CAV1 knockdown decreases BMPR2 localization at the plasma membrane and Smad 1/5/9 phosphorylation. hPAECs were transfected with control or CAV1-specific siRNA. a The BMP/TGF-β signaling pathway was assessed by western blotting. *P < 0.05 compared to control siRNA. ***P < 0.001 compared with control siRNA. ns, not significant. b Membrane and cytosol fractions were separated by sucrose gradient, and lysates of these fractions were immunoblotted. GAPDH was used as a cytosolic marker and VE-cadherin was used as a membrane marker. *P < 0.05 compared to control siRNA. c hPAECs transfected with control or CAV1-specific siRNA were immunostained with anti-VE-cadherin and anti-BMPR2 antibodies. Higher-magnification images are shown on the upper right. Scale bar, 100 μm. Data are expressed as the mean ± standard error.

Article Snippet: The rabbit polyclonal anti-CAV1 antibody (#sc-894), the mouse monoclonal anti-CAV1 antibody (#sc-70516), the monoclonal anti-BMPR1a antibody (#sc-518037) and the mouse monoclonal anti-VE cadherin antibody (#sc-9989) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); the rabbit polyclonal antibody to Cavin-1 (#ab76919) and αSMA (#ab124964), the horseradish peroxidase-conjugated monoclonal antibody to GAPDH (#ab105428) and the mouse monoclonal antibody to myc tag (#ab32) were from Abcam PLC (Cambridge, UK); the rabbit polyclonal antibodies to Akt (#9272), phosphoAkt (Ser473) (#9271), phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad9 (Ser465/467) (#13820), Smad1 (#9743), phospho-Smad2 (Ser465/467) (#3108), Smad2/3 (#3102) and the horseradish peroxidase-conjugated secondary antibodies (antimouse-HRP, and anti-rabbit-HRP) were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit polyclonal antibody to Cavin-2 (#12339-1-AP) and Cavin-3 (PRKCDBP) (#16250-1-AP) was purchased from ProteinTech Group, Inc. (Rosemont, IL, USA); the rabbit polyclonal anti-Cavin-4 antibody was generated as previously described58,59; the mouse monoclonal antibody to FLAG (clone M2, #F3165,), the mouse monoclonal antibody to T7 (#69522), Cy3-conjugated monoclonal antibody to αSMA (#C6198) and collagenase type 1 from Clostridium histolyticum were from Merck (Darmstadt, Germany); the mouse monoclonal antibodies to CAV3 (#610421) and the rat monoclonal antibodies to CD31 (clone MEC13.3, #550274) were from BD Biosciences (Franklin Lakes, NJ, USA); the horseradish peroxidase-conjugated monoclonal antibody to GST (#NB600-388) was from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Knockdown, Clinical Proteomics, Membrane, Phospho-proteomics, Transfection, Control, Western Blot, Marker

Journal: Communications biology

Article Title: The Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling in pulmonary hypertension by interfering with BMPR2/Caveolin-1 binding.

doi: 10.1038/s42003-023-05693-2

Figure Lengend Snippet: Fig. 7 Working hypothesis. Proposed roles of Cavin-1 include competitively inhibiting the binding of CAV1 and BMPR2 in the caveolin scaffolding domain and attenuating BMPR2 localization at the plasma membrane and BMP/Smad signaling in the progression of PAH. CSD caveolin scaffolding domain.

Article Snippet: The rabbit polyclonal anti-CAV1 antibody (#sc-894), the mouse monoclonal anti-CAV1 antibody (#sc-70516), the monoclonal anti-BMPR1a antibody (#sc-518037) and the mouse monoclonal anti-VE cadherin antibody (#sc-9989) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); the rabbit polyclonal antibody to Cavin-1 (#ab76919) and αSMA (#ab124964), the horseradish peroxidase-conjugated monoclonal antibody to GAPDH (#ab105428) and the mouse monoclonal antibody to myc tag (#ab32) were from Abcam PLC (Cambridge, UK); the rabbit polyclonal antibodies to Akt (#9272), phosphoAkt (Ser473) (#9271), phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad9 (Ser465/467) (#13820), Smad1 (#9743), phospho-Smad2 (Ser465/467) (#3108), Smad2/3 (#3102) and the horseradish peroxidase-conjugated secondary antibodies (antimouse-HRP, and anti-rabbit-HRP) were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit polyclonal antibody to Cavin-2 (#12339-1-AP) and Cavin-3 (PRKCDBP) (#16250-1-AP) was purchased from ProteinTech Group, Inc. (Rosemont, IL, USA); the rabbit polyclonal anti-Cavin-4 antibody was generated as previously described58,59; the mouse monoclonal antibody to FLAG (clone M2, #F3165,), the mouse monoclonal antibody to T7 (#69522), Cy3-conjugated monoclonal antibody to αSMA (#C6198) and collagenase type 1 from Clostridium histolyticum were from Merck (Darmstadt, Germany); the mouse monoclonal antibodies to CAV3 (#610421) and the rat monoclonal antibodies to CD31 (clone MEC13.3, #550274) were from BD Biosciences (Franklin Lakes, NJ, USA); the horseradish peroxidase-conjugated monoclonal antibody to GST (#NB600-388) was from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Binding Assay, Scaffolding, Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 reduces CAV1/E-cad complex formation and augments phosphorylation of CAV1 on tyrosine-14 in B16F10 cells. B16F10 (mock) and B16F10 (CAV1) cells were cultured in the presence of IPTG (1 mM) and then transiently transfected (or not) with pBATEM to overexpress E-cad. After 48 h, cell extracts from B16F10 (mock), B16F10 (CAV1), B16F10 (mock/E-cad), and B16F10 (CAV1/E-cad) cells were separated using SDS-PAGE in 12% minigels (50 μg total protein per lane). CAV1 and E-cad levels were evaluated via Western blotting using specific antibodies. Actin was used as a loading control. ( A ) Results from a representative immunoblot experiment. CAV1 ( B ) and E-cad ( C ) protein levels were quantified in several experiments via scanning densitometry and normalized to actin. ( D ) B16F10 (mock/E-cad) or B16F10 (CAV1/E-cad) cells were serum deprived for 5 h and treated or not with PGE2 (20 μM) for 2 h. Then, CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via immunoblotting. The left panel shows a representative result, and the blot on the right shows the total protein input of each sample. ( E ) The values in the graph show the CAV1/E-cad ratios normalized to actin. ( F ) Protein extracts from B16F10 (mock) and B16F10 (CAV1) cells treated with 20 μM PGE2 or vehicle (control) for 2 h. Phospho-CAV1 (pY14-CAV1) and CAV1 and E-cad levels were evaluated via immunoblotting. Results from a representative experiment are shown. ( G ) pY14-CAV1, CAV1, and E-cad levels were quantified via scanning densitometry and are shown as the pY14-CAV1/CAV1 ratio standardized to mock (−) controls. Values in all graphs are the mean ± SD, n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Cell Culture, Transfection, SDS Page, Western Blot, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 enhances migration and invasion of B16F10 (CAV1/E-cad) cells. B16F10 (mock) and B16F10 (CAV1) cells cultured in the presence of IPTG (1 mM) were transiently transfected with pBATEM to overexpress E-cad. After 48 h, B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), and B16F10 (CAV1/E-cad) cultures were treated with 20 μM PGE2 for another 24 h, and then cell migration and invasion were evaluated. ( A ) B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad) cells (150,000) treated or not with PGE2 were seeded in Boyden chambers (transwells) in the presence or absence of 20 μM PGE2 and allowed to transmigrate for 16 h. The graph shows the fold change in the migration of cells treated or not with PGE2, normalized to B16F10 (mock) control cells. Significant differences are shown (* p < 0.05, n = 3). ( B ) The panels show representative images from the migration assays of cells in the absence ( upper panel ) or presence ( lower panel ) of PGE2. Magnification bar = 50 µm. ( C ) Cells treated or not with PGE2 (20 µM) for 24 h were used for the invasion assays. Cells (150,000) were seeded in matrigel Boyden chambers in the absence or presence of PGE2 (20 μM). Cell invasion was measured after 24 h. The graph shows the fold change in invasion normalized to B16F10 (mock) control cells. Significant differences are shown (* p < 0.05; n = 3). ( D ) The panel shows representative images of the invasion assays in the absence ( upper panel ) or presence ( lower panel ) of PGE2. Magnification bar = 50 μm.

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Migration, Cell Culture, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 enhances subcutaneous tumor formation and lung metastasis of B16F10 (CAV1/E-cad) cells. ( A ) Scheme illustrating experimental procedures in tumor formation assays. C57BL/6 mice between 5 and 7 weeks of age were used for these assays. Mice were injected subcutaneously (day 0) with B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad) cells (1 × 10 5 ), pre-treated or not with 20 μM of PGE2 (for 24 h). At different time intervals, up to 29 days, tumor size was evaluated in all experimental groups, as indicated in the formula. ( B.1 – B.3 ) Graph showing the tumor volume (mm 3 ) for animals at different time points post-injection of cells pre-treated or not with PGE2, and the cells injected were B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad). The results are shown as the mean ± SEM (n = 3; ** p < 0.01). ( B.1 ) Graph showing a time-course experiment for mice injected with cells that were not previously incubated with PGE2. ( B.2 ) Graph of time-course experiment for mice injected with cells that were previously incubated with PGE2. ( B.3 ) Graph showing the curves for all time-course experiments. The details concerning the number of mice employed are described in the methodology section. ( C ) A scheme illustrating the experimental procedures in metastasis assays. Cells in culture pre-treated or not with 20 μM of PGE2 for 24 h were harvested, re-suspended in physiological serum, and then mice were injected intravenously in the tail vein with 2 × 10 5 of B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad) cells (2 × 10 5 ). After 21 days, the mice were euthanized and necropsied. The black lung tumor mass was quantified. ( D ) The graph shows lung tumor mass (g) in different experimental animal groups, and representative images of tumor nodules in the lungs are shown; melanoma nodules are observed as black tissue. Results are shown as the mean ± SEM (n = 3; ** p < 0.01).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Injection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 increases migration and invasion of A375M (CAV1/E-cad) cells. A375 (Mock) and A375 (CAV1) cell lines cultured in the presence of IPTG were transiently transfected with pBATEM for 48 h to express E-cad. A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells were treated with 50 μM of PGE2 for 5 h. Then, the extracts were either prepared for immunoprecipitation experiments or cell migration and invasion measurements in transwell and matrigel assays, respectively. ( A ) CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Representative Western blot of immunoprecipitates obtained in three independent experiments. E-cad and CAV1 protein levels were quantified in several experiments via densitometry and normalized to actin (mean ± SD, n = 3; ** p < 0.01). A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were serum-deprived for 5 h and treated with PGE2 (50 μM) for 2 h. Then, CAV1 was immunoprecipitated from the protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Graph showing the CAV1 as normalized pixels (mean ± SEM) for each condition; n = 3; and ** p < 0.01 with respect to A375 (mock). ( C ) Graph showing E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; ** p < 0.01 with respect to A375 (mock). ( D ) CAV1/E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; and * p < 0.05 or ** p < 0.01 with respect to A375 (mock). ( E ) A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were treated with 50 μM of PGE2 for 5 h. Then, 150.000 cells were seeded into Boyden chambers (transwells) in the presence of 50 μM PGE2. Migration was measured after 2 h. Significant differences are shown (* p < 0.05; n = 3). ( F ) Panels showing representative images of results obtained for cells migrating either without treatment (basal condition, upper panel ) or when treated with 50 μM of PGE2 for 5 h ( lower panel ), in both cases stained with crystal violet. ( H ) Cells cultured in the presence or absence of 50 µM of PGE2 for 5 h were used for the invasion assays. Cells (150.000) were seeded into matrigel Boyden chambers in the presence of 50 μM of PGE2. Invasion was measured after 48 h. Graphs show averages (mean ± SEM; n = 3). Significant differences are indicated (* p < 0.05). ( G ) Panel showing representative images obtained in invasion assays for cells cultured in the absence (basal condition, upper panel ) or presence ( lower panel ) of 50 μM of PGE2, in both cases stained with DAPI Bar = 50 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Migration, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 increases colony formation by A375 (CAV1/E-cad) cells. ( A ) For the clonogenic assays, A375 (mock), A375 (CAV1), A375 (E-cad), and A375 (E-cadCAV1/E-cad) cells were previously treated for 5 h with 50 μM of PGE2. Cells (3 × 106) were mixed with 0.2% agarose and plated on low-adhesion plates for 24 h. Subsequently, cells were treated with PGE2 (diluted in medium supplemented with 10% FBS) for 14 days. Representative images of A375 cells treated ( upper panel ) or not treated ( lower panel ) with PGE2 are shown. The size of cell clusters is indicated in each image, in black arrows showed the clones, and in white line is the measured representative of clone. Colonies were then counted, but only colonies larger than 100 μm were considered. ( B ) Graph showing the number of cell colonies formed by A375 (mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells treated or not with 50 μM PGE2. Results shown are the mean ± SEM (n = 3; *** p < 0.001).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 enhances spleen and abdominal metastasis of A375 (CAV1/E-cad) cells. ( A ) Scheme showing the time course of the intraperitoneal carcinomatosis assays. Pathogen-free immunodeficient B16Rag1−/− mice between 5 and 7 weeks of age were used for the metastasis assays. Cells in culture were harvested, re-suspended in physiological serum, and pre-treated or not with 50 μM of PGE2 for 5 h. Then, 2.5 × 10 6 A375 (mock), A375 (CAV1), A375 (E-cad), or A375 (CAV1/E-cad) cells were injected into the peritoneal cavity of the mice (day 0). After 21 days, all animals were necropsied. The total spleen mass was visualized and quantified. ( B ) Representative images of the total spleen mass (which includes the spleen and tissue-adherent tumor mass). ( C ) The graph shows the average total mass of spleens (g) in mice (mean ± SEM; n = 5). ( D ) The solid tumor mass in the abdominal cavity was also quantified. The graph shows the average abdominal tumor mass (g) in animals (mean ± SEM) from five mice (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: Summary model illustrating the effect of PGE2 on the CAV1/E-cad complex.

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 reduces CAV1/E-cad complex formation and augments phosphorylation of CAV1 on tyrosine-14 in B16F10 cells. B16F10 (mock) and B16F10 (CAV1) cells were cultured in the presence of IPTG (1 mM) and then transiently transfected (or not) with pBATEM to overexpress E-cad. After 48 h, cell extracts from B16F10 (mock), B16F10 (CAV1), B16F10 (mock/E-cad), and B16F10 (CAV1/E-cad) cells were separated using SDS-PAGE in 12% minigels (50 μg total protein per lane). CAV1 and E-cad levels were evaluated via Western blotting using specific antibodies. Actin was used as a loading control. ( A ) Results from a representative immunoblot experiment. CAV1 ( B ) and E-cad ( C ) protein levels were quantified in several experiments via scanning densitometry and normalized to actin. ( D ) B16F10 (mock/E-cad) or B16F10 (CAV1/E-cad) cells were serum deprived for 5 h and treated or not with PGE2 (20 μM) for 2 h. Then, CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via immunoblotting. The left panel shows a representative result, and the blot on the right shows the total protein input of each sample. ( E ) The values in the graph show the CAV1/E-cad ratios normalized to actin. ( F ) Protein extracts from B16F10 (mock) and B16F10 (CAV1) cells treated with 20 μM PGE2 or vehicle (control) for 2 h. Phospho-CAV1 (pY14-CAV1) and CAV1 and E-cad levels were evaluated via immunoblotting. Results from a representative experiment are shown. ( G ) pY14-CAV1, CAV1, and E-cad levels were quantified via scanning densitometry and are shown as the pY14-CAV1/CAV1 ratio standardized to mock (−) controls. Values in all graphs are the mean ± SD, n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Cell Culture, Transfection, SDS Page, Western Blot, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 enhances migration and invasion of B16F10 (CAV1/E-cad) cells. B16F10 (mock) and B16F10 (CAV1) cells cultured in the presence of IPTG (1 mM) were transiently transfected with pBATEM to overexpress E-cad. After 48 h, B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), and B16F10 (CAV1/E-cad) cultures were treated with 20 μM PGE2 for another 24 h, and then cell migration and invasion were evaluated. ( A ) B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad) cells (150,000) treated or not with PGE2 were seeded in Boyden chambers (transwells) in the presence or absence of 20 μM PGE2 and allowed to transmigrate for 16 h. The graph shows the fold change in the migration of cells treated or not with PGE2, normalized to B16F10 (mock) control cells. Significant differences are shown (* p < 0.05, n = 3). ( B ) The panels show representative images from the migration assays of cells in the absence ( upper panel ) or presence ( lower panel ) of PGE2. Magnification bar = 50 µm. ( C ) Cells treated or not with PGE2 (20 µM) for 24 h were used for the invasion assays. Cells (150,000) were seeded in matrigel Boyden chambers in the absence or presence of PGE2 (20 μM). Cell invasion was measured after 24 h. The graph shows the fold change in invasion normalized to B16F10 (mock) control cells. Significant differences are shown (* p < 0.05; n = 3). ( D ) The panel shows representative images of the invasion assays in the absence ( upper panel ) or presence ( lower panel ) of PGE2. Magnification bar = 50 μm.

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Migration, Cell Culture, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 enhances subcutaneous tumor formation and lung metastasis of B16F10 (CAV1/E-cad) cells. ( A ) Scheme illustrating experimental procedures in tumor formation assays. C57BL/6 mice between 5 and 7 weeks of age were used for these assays. Mice were injected subcutaneously (day 0) with B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad) cells (1 × 10 5 ), pre-treated or not with 20 μM of PGE2 (for 24 h). At different time intervals, up to 29 days, tumor size was evaluated in all experimental groups, as indicated in the formula. ( B.1 – B.3 ) Graph showing the tumor volume (mm 3 ) for animals at different time points post-injection of cells pre-treated or not with PGE2, and the cells injected were B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad). The results are shown as the mean ± SEM (n = 3; ** p < 0.01). ( B.1 ) Graph showing a time-course experiment for mice injected with cells that were not previously incubated with PGE2. ( B.2 ) Graph of time-course experiment for mice injected with cells that were previously incubated with PGE2. ( B.3 ) Graph showing the curves for all time-course experiments. The details concerning the number of mice employed are described in the methodology section. ( C ) A scheme illustrating the experimental procedures in metastasis assays. Cells in culture pre-treated or not with 20 μM of PGE2 for 24 h were harvested, re-suspended in physiological serum, and then mice were injected intravenously in the tail vein with 2 × 10 5 of B16F10 (mock), B16F10 (CAV1), B16F10 (E-cad), or B16F10 (CAV1/E-cad) cells (2 × 10 5 ). After 21 days, the mice were euthanized and necropsied. The black lung tumor mass was quantified. ( D ) The graph shows lung tumor mass (g) in different experimental animal groups, and representative images of tumor nodules in the lungs are shown; melanoma nodules are observed as black tissue. Results are shown as the mean ± SEM (n = 3; ** p < 0.01).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Injection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 increases migration and invasion of A375M (CAV1/E-cad) cells. A375 (Mock) and A375 (CAV1) cell lines cultured in the presence of IPTG were transiently transfected with pBATEM for 48 h to express E-cad. A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells were treated with 50 μM of PGE2 for 5 h. Then, the extracts were either prepared for immunoprecipitation experiments or cell migration and invasion measurements in transwell and matrigel assays, respectively. ( A ) CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Representative Western blot of immunoprecipitates obtained in three independent experiments. E-cad and CAV1 protein levels were quantified in several experiments via densitometry and normalized to actin (mean ± SD, n = 3; ** p < 0.01). A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were serum-deprived for 5 h and treated with PGE2 (50 μM) for 2 h. Then, CAV1 was immunoprecipitated from the protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Graph showing the CAV1 as normalized pixels (mean ± SEM) for each condition; n = 3; and ** p < 0.01 with respect to A375 (mock). ( C ) Graph showing E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; ** p < 0.01 with respect to A375 (mock). ( D ) CAV1/E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; and * p < 0.05 or ** p < 0.01 with respect to A375 (mock). ( E ) A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were treated with 50 μM of PGE2 for 5 h. Then, 150.000 cells were seeded into Boyden chambers (transwells) in the presence of 50 μM PGE2. Migration was measured after 2 h. Significant differences are shown (* p < 0.05; n = 3). ( F ) Panels showing representative images of results obtained for cells migrating either without treatment (basal condition, upper panel ) or when treated with 50 μM of PGE2 for 5 h ( lower panel ), in both cases stained with crystal violet. ( H ) Cells cultured in the presence or absence of 50 µM of PGE2 for 5 h were used for the invasion assays. Cells (150.000) were seeded into matrigel Boyden chambers in the presence of 50 μM of PGE2. Invasion was measured after 48 h. Graphs show averages (mean ± SEM; n = 3). Significant differences are indicated (* p < 0.05). ( G ) Panel showing representative images obtained in invasion assays for cells cultured in the absence (basal condition, upper panel ) or presence ( lower panel ) of 50 μM of PGE2, in both cases stained with DAPI Bar = 50 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Migration, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 increases colony formation by A375 (CAV1/E-cad) cells. ( A ) For the clonogenic assays, A375 (mock), A375 (CAV1), A375 (E-cad), and A375 (E-cadCAV1/E-cad) cells were previously treated for 5 h with 50 μM of PGE2. Cells (3 × 106) were mixed with 0.2% agarose and plated on low-adhesion plates for 24 h. Subsequently, cells were treated with PGE2 (diluted in medium supplemented with 10% FBS) for 14 days. Representative images of A375 cells treated ( upper panel ) or not treated ( lower panel ) with PGE2 are shown. The size of cell clusters is indicated in each image, in black arrows showed the clones, and in white line is the measured representative of clone. Colonies were then counted, but only colonies larger than 100 μm were considered. ( B ) Graph showing the number of cell colonies formed by A375 (mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells treated or not with 50 μM PGE2. Results shown are the mean ± SEM (n = 3; *** p < 0.001).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 enhances spleen and abdominal metastasis of A375 (CAV1/E-cad) cells. ( A ) Scheme showing the time course of the intraperitoneal carcinomatosis assays. Pathogen-free immunodeficient B16Rag1−/− mice between 5 and 7 weeks of age were used for the metastasis assays. Cells in culture were harvested, re-suspended in physiological serum, and pre-treated or not with 50 μM of PGE2 for 5 h. Then, 2.5 × 10 6 A375 (mock), A375 (CAV1), A375 (E-cad), or A375 (CAV1/E-cad) cells were injected into the peritoneal cavity of the mice (day 0). After 21 days, all animals were necropsied. The total spleen mass was visualized and quantified. ( B ) Representative images of the total spleen mass (which includes the spleen and tissue-adherent tumor mass). ( C ) The graph shows the average total mass of spleens (g) in mice (mean ± SEM; n = 5). ( D ) The solid tumor mass in the abdominal cavity was also quantified. The graph shows the average abdominal tumor mass (g) in animals (mean ± SEM) from five mice (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: Summary model illustrating the effect of PGE2 on the CAV1/E-cad complex.

Article Snippet: Rabbit polyclonal anti-CAV1 (ab18199 rabbit polyclonal), mouse monoclonal anti-CAV1 (C13620), mouse monoclonal anti-pY14-CAV1 (C611339) and mouse monoclonal anti-E-cad (C61018) antibodies were obtained from BD Transduction Laboratories, NJ, USA; monoclonal anti-β-actin antibodies (sc47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and all were used as indicated by the manufacturers.

Techniques:

Journal: Nature cell biology

Article Title: Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

doi: 10.1038/s41556-022-01034-3

Figure Lengend Snippet: Fig. 1 | Cav1 confers protection to hypo-osmotic treatment. a–c, Schematic representations of the different caveolae-related phenotypes. d–f, EM images of PM regions from MEFs, showing the presence of caveolae exclusively in PTRFKO MEFs reconstituted with PTRF (f, black arrows). g, Percentage of dead cells after hypo-osmotic shock (fragility assay in suspension; for details, see Methods) across indicated genotypes. Data are presented as mean ± s.e.m. n = 32 independent fragility assays. Statistical comparisons were by two-tailed Student’s t-test (comparing PTRFKO with either PTRFKO + Cav1 high expression, P = 0.0195; PTRFKO + PTRF, P = 0.0434; or MEFs WT, P = 0.0048), with significance assigned at *P < 0.05. Source numerical data are available in source data.

Article Snippet: The following primary antibodies were used: rat monoclonal anti-mouse total beta 1 integrin (clone MB1.2, MAB1997 Millipore, 1:1,000); rabbit monoclonal anti-mouse Cav1 (Cell Signaling, 1:1,000 for western blot and 1:100 for immunofluorescence); rabbit polyclonal anti-mouse PTRF (ab48824, Abcam, 1:1,000 for western blot and 1:100 for immunofluorescence); mouse monoclonal anti-alpha tubulin (ab7291, Abcam, 1:10,000); anti-Cav1 N-20 antibody (Santa Cruz Sc-894); Cav1 SIGMA SAB4200216 (mouse monoclonal); ubiquitin (Enzo, ENZ-ABS840-0100); mouse monoclonal anti-GAPDH (sc47724, Santa Cruz, 1:1,000); and mouse anti-GFP (catalogue number 118114460001, Roche, 1:1,000).

Techniques: Suspension, Two Tailed Test, Expressing

Journal: Nature cell biology

Article Title: Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

doi: 10.1038/s41556-022-01034-3

Figure Lengend Snippet: Fig. 3 | Cav1 buffers tension in the absence of caveolae. a, OT experiment scheme, indicating the cell, the OT beam, the cellular nanotube and the bead attached to the cell surface. b, Relative change of the mean tether force after hypo-osmotic shock (150 mOsm) for PTRFKO MEFs reconstituted with PTRF (n = 9), Cav1 (n = 10) or empty vector (n = 14). n indicates number of cells pooled from eight independent experiments. c, Relative change of the mean tether force after hypo-osmotic shock (60 mOsm) as a function of GFP intensity (which correlates with Cav1 levels, Extended Data Fig. 1; a. u., arbitrary units) of PTRFKO reconstituted with Cav1 (n = 33). n indicates number of cells pooled from six independent experiments. For b and c, individual values are plotted (data are presented as mean ± s.e.m.), statistical analysis strategy used was one-way ANOVA with Tukey’s multiple comparisons test, with significance assigned at *P < 0.01 and ***P < 0.0003, NS, non-significant. Source numerical data are available in source data.

Article Snippet: The following primary antibodies were used: rat monoclonal anti-mouse total beta 1 integrin (clone MB1.2, MAB1997 Millipore, 1:1,000); rabbit monoclonal anti-mouse Cav1 (Cell Signaling, 1:1,000 for western blot and 1:100 for immunofluorescence); rabbit polyclonal anti-mouse PTRF (ab48824, Abcam, 1:1,000 for western blot and 1:100 for immunofluorescence); mouse monoclonal anti-alpha tubulin (ab7291, Abcam, 1:10,000); anti-Cav1 N-20 antibody (Santa Cruz Sc-894); Cav1 SIGMA SAB4200216 (mouse monoclonal); ubiquitin (Enzo, ENZ-ABS840-0100); mouse monoclonal anti-GAPDH (sc47724, Santa Cruz, 1:1,000); and mouse anti-GFP (catalogue number 118114460001, Roche, 1:1,000).

Techniques: Plasmid Preparation

Journal: Nature cell biology

Article Title: Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

doi: 10.1038/s41556-022-01034-3

Figure Lengend Snippet: Fig. 4 | Cav1 forms clusters of different sizes in the absence of PTRF. a–c, dSTORM representative images of MEFs of the indicated genotypes (left) and analyses of clusters showing the size and separation of nanostructures (right). These plots were taken from the corresponding dSTORM images and show the cutting curves (green) for profile analysis and full width at half maximum (FWHM) for each fitted peak in representative clusters. Scale bar, 2 µm; a.u., arbitrary units. Results are representative of 6 WT, 10 PTRFKO + PTRF and 11 PTRFKO + Cav1 cells from two independent replicates. d, Cluster density expressed as percentage area for the different MEF genotypes. e, Normalized cluster frequency segmented according to the Feret diameter (longest distance between any two points along the selection boundary44; Results) of

Article Snippet: The following primary antibodies were used: rat monoclonal anti-mouse total beta 1 integrin (clone MB1.2, MAB1997 Millipore, 1:1,000); rabbit monoclonal anti-mouse Cav1 (Cell Signaling, 1:1,000 for western blot and 1:100 for immunofluorescence); rabbit polyclonal anti-mouse PTRF (ab48824, Abcam, 1:1,000 for western blot and 1:100 for immunofluorescence); mouse monoclonal anti-alpha tubulin (ab7291, Abcam, 1:10,000); anti-Cav1 N-20 antibody (Santa Cruz Sc-894); Cav1 SIGMA SAB4200216 (mouse monoclonal); ubiquitin (Enzo, ENZ-ABS840-0100); mouse monoclonal anti-GAPDH (sc47724, Santa Cruz, 1:1,000); and mouse anti-GFP (catalogue number 118114460001, Roche, 1:1,000).

Techniques: Selection

Journal: Nature cell biology

Article Title: Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

doi: 10.1038/s41556-022-01034-3

Figure Lengend Snippet: Fig. 5 | PTRF genetic ablation leads to loss of caveolae and accumulation of non-caveolar Cav1 immunopositive structures. a–d, Anti-Cav1 immunogold-labelled PMs of WT (a), PTRFKO (b), PTRFKO + Cav1 (c) and PTRFKO + PTRF MEFs (d). Caveolae (a, arrows) and anti-Cav1-positive shallow invaginations (a, arrowheads) were absent in b and c. Scale bars, 100 nm. e, Anti-Cav1 immunolabelling density across all four MEFs genotypes. f,g, Caveolae densities (f, deep; g, shallow). h,i, Densities of ‘clustered’ (h) and ‘disperse’ (i) anti-Cav1 immunogold labels. j,k, Cav1-immunopositive non-caveolar structures (red arrowheads) in PTRFKO + Cav1 MEFs (j and j′], morphologically distinct from caveolae, are also found (usually smaller) in WT cells (k). Scale bars, 100 nm. l–o, Electron tomograms (l and n) and 3D reconstructions (m and o) of a non-caveolar structure at the PM of PTRFKO + Cav1 cells (l and m), and ‘classical’ caveolae in PTRFKO + PTRF cells (n and o). Marks as in a, j, j′ and k, respectively. In m and o, gold particles: yellow; PM: green. Scale bars, 200 nm. p, Density distribution of non-caveolar anti-Cav1-positive structures in PRTFKO + PTRF and WT cells (grey shadowed area; diameters up to 299 nm);

Article Snippet: The following primary antibodies were used: rat monoclonal anti-mouse total beta 1 integrin (clone MB1.2, MAB1997 Millipore, 1:1,000); rabbit monoclonal anti-mouse Cav1 (Cell Signaling, 1:1,000 for western blot and 1:100 for immunofluorescence); rabbit polyclonal anti-mouse PTRF (ab48824, Abcam, 1:1,000 for western blot and 1:100 for immunofluorescence); mouse monoclonal anti-alpha tubulin (ab7291, Abcam, 1:10,000); anti-Cav1 N-20 antibody (Santa Cruz Sc-894); Cav1 SIGMA SAB4200216 (mouse monoclonal); ubiquitin (Enzo, ENZ-ABS840-0100); mouse monoclonal anti-GAPDH (sc47724, Santa Cruz, 1:1,000); and mouse anti-GFP (catalogue number 118114460001, Roche, 1:1,000).

Techniques:

Journal: Nature cell biology

Article Title: Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

doi: 10.1038/s41556-022-01034-3

Figure Lengend Snippet: Fig. 6 | Dolines and caveolae respond differently to membrane tension. a, Formation and tension-induced disassembly of protein-rich curved domains according to theoretical model; protein distribution: colour map depicting protein area fraction. Spontaneous curvature is smaller in Cav1 as compared with PTRF model, leading to shallow caps and full buds respectively when tension is low (a1 and b1). The Cav1 model exhibits gradual and reversible disassembly whereas for the PTRF model it is sudden and hysteretic. b, Theoretical model to understand the behaviour of an ensemble of such domains (Methods). c, Predictions of this model for the projected diameter of each domain, density of domains and projected areal strain of the membrane as a function of applied tension for three different average protein area fractions. Rightmost panels show representative states as tension varies for each model. d, Percentage of dead cells upon increasing medium dilution and hypo-osmotic shock (1 min fragility assay in adhesion) across indicated genotypes. Plots: mean ± s.e.m. n = 20 independent assays. Statistics: one-way ANOVA with Bonferroni post-test (P = 0.0484 at 1/4 dilution and P = 0.0324at 1/20 dilution). e, YAP activation as

Article Snippet: The following primary antibodies were used: rat monoclonal anti-mouse total beta 1 integrin (clone MB1.2, MAB1997 Millipore, 1:1,000); rabbit monoclonal anti-mouse Cav1 (Cell Signaling, 1:1,000 for western blot and 1:100 for immunofluorescence); rabbit polyclonal anti-mouse PTRF (ab48824, Abcam, 1:1,000 for western blot and 1:100 for immunofluorescence); mouse monoclonal anti-alpha tubulin (ab7291, Abcam, 1:10,000); anti-Cav1 N-20 antibody (Santa Cruz Sc-894); Cav1 SIGMA SAB4200216 (mouse monoclonal); ubiquitin (Enzo, ENZ-ABS840-0100); mouse monoclonal anti-GAPDH (sc47724, Santa Cruz, 1:1,000); and mouse anti-GFP (catalogue number 118114460001, Roche, 1:1,000).

Techniques: Membrane, Activation Assay

Journal: Nature cell biology

Article Title: Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

doi: 10.1038/s41556-022-01034-3

Figure Lengend Snippet: Fig. 7 | Characterization of dolines and working model description. a,b, TIRF microscopy snapshots of Cav1GFP cluster formation (a), comparing PTRFKO MEFs co-electroporated with either Cav1GFP and empty vector (Cav1 alone cells) or Cav1GFP and PTRF vectors (control cells) (b). Mean ± s.e.m. n = 13 independent fields pooling together three cells (Cav1 alone); and n = 15 independent fields pooling together four cells (control). Two-tailed Student’s t-test (P = 0.0317). c,d, Maximal projection of TIRF microscopy image series (from Supplementary Videos 13 and 14) of Cav1GFP clusters from Cav1 alone (left) or control (right) PTRFKO MEFs (see a and b), after treatment with methyl-β-cyclodextrin. Time lapse of a splitting cluster shown below. In d, mean ± s.e.m. n = 22 independent experiments pooling together 22 cells (Cav1 alone); and n = 20 independent experiments pooling together 20 cells (control), after treatment with methyl-β-cyclodextrin. Two-tailed Student’s t-test (P = 0.0221). e, Cluster densities across genotypes and osmotic conditions from dSTORM imaging, in three size groups. Data points outside the whiskers are considered outliers and plotted as empty circles. Boxes span Q1 to Q3 quartiles,

Article Snippet: The following primary antibodies were used: rat monoclonal anti-mouse total beta 1 integrin (clone MB1.2, MAB1997 Millipore, 1:1,000); rabbit monoclonal anti-mouse Cav1 (Cell Signaling, 1:1,000 for western blot and 1:100 for immunofluorescence); rabbit polyclonal anti-mouse PTRF (ab48824, Abcam, 1:1,000 for western blot and 1:100 for immunofluorescence); mouse monoclonal anti-alpha tubulin (ab7291, Abcam, 1:10,000); anti-Cav1 N-20 antibody (Santa Cruz Sc-894); Cav1 SIGMA SAB4200216 (mouse monoclonal); ubiquitin (Enzo, ENZ-ABS840-0100); mouse monoclonal anti-GAPDH (sc47724, Santa Cruz, 1:1,000); and mouse anti-GFP (catalogue number 118114460001, Roche, 1:1,000).

Techniques: Microscopy, Plasmid Preparation, Control, Two Tailed Test, Imaging