Name: rabbit anti-brca1 polyclonal antibody (cat no. ptg-22363-1-ap)
Brand: Millennium Science
Rating: 90 (1 reviews)

rabbit anti-brca1 polyclonal antibody (cat no. ptg-22363-1-ap) (Millennium Science)

Millennium Science rabbit anti-brca1 polyclonal antibody (cat no. ptg-22363-1-ap)

( A ). Z-score for HeLa WT and cavin3 KO cells (Replicates Rep. 1-3) showing upregulated proteins (red) and downregulated proteins (blue). ( B ). Volcano plot showing proteins (red dots) identified by GOBP involved in DNA repair. ( C ). Volcano plot showing DNA repair proteins upregulated in cavin3 KO cells. ( D ). Volcano plot showing proteins of the <t>BRCA1</t> A-complex, BRCA1, BRCC3, MDC1 and UBE4A downregulated in cavin3 HeLa KO cells and upregulation of TP53BP1 with a heatmap analysis of the expression of each of these proteins in Replicate (Rep. 1-3) HeLa WT and cav in3 KO cells.

Rabbit Anti Brca1 Polyclonal Antibody (Cat No. Ptg 22363 1 Ap), supplied by Millennium Science, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: ( A ). Z-score for HeLa WT and cavin3 KO cells (Replicates Rep. 1-3) showing upregulated proteins (red) and downregulated proteins (blue). ( B ). Volcano plot showing proteins (red dots) identified by GOBP involved in DNA repair. ( C ). Volcano plot showing DNA repair proteins upregulated in cavin3 KO cells. ( D ). Volcano plot showing proteins of the BRCA1 A-complex, BRCA1, BRCC3, MDC1 and UBE4A downregulated in cavin3 HeLa KO cells and upregulation of TP53BP1 with a heatmap analysis of the expression of each of these proteins in Replicate (Rep. 1-3) HeLa WT and cav in3 KO cells.

Article Snippet: The following antibodies were used: rabbit anti-CAV1 (Cat no. 610060, BD Biosciences, Franklin Lakes, New Jersey, USA), rabbit anti-cavin1 antibody were raised as described previously and was used for immunofluorescence , rabbit anti-cavin3 (Cat no. 16250-1-AP, 1:2000 WB, 1:300 PLA, Millennium Science, Pty, Ltd), mouse anti-cavin1 (1:100 PLA, Abmart, China), rabbit anti-cavin1 (Cat no. AV36965, 1:2000 WB, Sigma Aldrich), mouse anti-cavin3 (Cat no. ABNOH00112464-MO4, 1:200 PLA, VWR International Pty, Australia), rabbit anti-BRCA1 C-20 (SC-642, 1:500 WB, 1:100 IF/PLA, Bio-Strategy Laboratory Products), mouse anti-BRCA1 MS110 (ab16780, 1:1000 WB, 1:100 IF/PLA, Abcam, Australia), rabbit anti-BRCA1 (07-434, 1:1000 WB, Merck Australia), mouse anti-BRCA1 antibody (D-9, SC-6954, 1:50 IF, Bio-Strategy Laboratory Products), rabbit anti-BRCA1 polyclonal antibody (Cat no. PTG-22363-1-AP, 1:1000 WB, Millennium Science, Pty, Ltd), mouse anti-BARD1 antibody (E-11) (Cat no. sc-74559, 1:500 WB, Bio-Strategy Laboratory Products), rabbit anti-BRCA2 antibody (Cat no. 3675-30T, WB 1: 2000, Sapphire Bioscience), mouse anti-GFP (Cat no. 11814460001, 1:300 PLA, 1:4000 WB, Roche Diagnostics), mouse anti-Tubulin (Cat no. AB7291, 1:3000, ABCAM Australia, Pty, Ltd), mouse anti-Flotillin-1 (Cat no. 610821, 1:100 PLA, BD Biosciences), mouse anti-PCNA (Cat no. NA03T, 1:100 PLA, Merck Millipore), mouse anti-Aurora kinase 1 (Cat no. 611082, 1:100 PLA, BD Biosciences), mouse anti-Actin (MAB1051, 1:5000 WB, Merck), rabbit anti-HLTF (Cat no. PTG-14286-1-AP, 1:2000 WB, Millennium Science), rabbit anti-RNF-168 (Cat no. GTX118147, GeneTex), rabbit anti-BRCC36 (Cat no 4311, 1:1000 WB, ProScience), rabbit anti-MDC1 (Cat no. NOVNB10056657SS, 1:100 WB, Novus), rabbit anti-RAP80 (Cat no.14466S, 1:1000 WB, 1:100 IF, D1T6Q rabbit mAb), mouse anti-Rad51 antibody (Cat no. 14B4, WB 1:1000, NOVNB10014825UL, Novus), sheep anti-Merit40 antibody (Cat no. RDSAF6604SP, WB 1:100, R and D systems), rabbit anti-PGK1 antibody (Cat no. GTX107614, WB 1:3000, Sapphire Bioscience), rabbit anti-PKM antibody (Cat no. GTX107977, WB 1:3000, Sapphire Bioscience), rabbit anti-Histone H2A.X-Chip Grade (Cat no. ab20669, 1:1000 WB, Abcam Australia), rabbit Phospho-Histone H2A.X (Ser 139) (20E3) (Cat no. 9718, 1:500 IF, Cell Signaling), anti-gamma H2AX phosphoS139 CHIP Grade (ab2893, WB: 1:3000, ABCAM).

Techniques: Expressing

(A). mRNA analysis of cavin3 in WT and cavin3 KO cells from three independent experiments performed in triplicate samples as Mean ± SD using student t-test, ** p<0.01. (B). Representative Western blot analysis of equally loaded lysates for cavin3, DDX21, ACLY, gamma-catenin, alpha-catenin, ACCA, EGFR, CAV1 and Actin in WT and cavin3 KO HeLa cell. (C). Western blot analysis of equally loaded lysates from WT, cavin3 KO and cavin3 KO transfected with cavin3 for cavin3, GFP, CAV1, Caldesmon, DDX21 and Tubulin as the loading control. Western blots are representative of three independent experiments. (D). Representative Western blot analysis of lysates from WT and cavin3 KO cells were Western blotted for cavin3, RAP80/UIM1C, BRCA1 Santa Cruz (SC), BRCA1 Proteintech (PT), BARD1, Merit40, MDC1, BRCC3, Rad51, BRCC45, TP53BP1, FANCD2, HLTF and Actin as the loading control.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). mRNA analysis of cavin3 in WT and cavin3 KO cells from three independent experiments performed in triplicate samples as Mean ± SD using student t-test, ** p<0.01. (B). Representative Western blot analysis of equally loaded lysates for cavin3, DDX21, ACLY, gamma-catenin, alpha-catenin, ACCA, EGFR, CAV1 and Actin in WT and cavin3 KO HeLa cell. (C). Western blot analysis of equally loaded lysates from WT, cavin3 KO and cavin3 KO transfected with cavin3 for cavin3, GFP, CAV1, Caldesmon, DDX21 and Tubulin as the loading control. Western blots are representative of three independent experiments. (D). Representative Western blot analysis of lysates from WT and cavin3 KO cells were Western blotted for cavin3, RAP80/UIM1C, BRCA1 Santa Cruz (SC), BRCA1 Proteintech (PT), BARD1, Merit40, MDC1, BRCC3, Rad51, BRCC45, TP53BP1, FANCD2, HLTF and Actin as the loading control.

Techniques: Western Blot, Transfection, Control

(A). Two-color single molecule fluorescence coincidence of BRCA1-GFP with (A). mCherry control, (B). mCherry-cavin1, (C). mCherry-cavin2, (D). mCherry-cavin3, (E). mCherry-CAV1 coexpressed in MCF-7 cells. The green curve represents BRCA1-GFP only events, the red curve represents mCherry only events and the yellow curve represents BRCA1-GFP + Cherry events. (F). Distribution of burst brightness measured for BRCA1-GFP (blue) and GFP control (green). (G). Schematic representation of domain organization of full-length wildtype (WT) BRCA1 and the truncated (Tr) 1-300 BRCA1 constructs. Nuclear export signal-NES, BRCA1 C Terminus domain (BRCT) domain, N-N Terminus (H-J). Two-color single molecule fluorescence coincidence of 1-300 BRCA1 with (H). cavin1, (I). cavin2 and (J). cavin3 expressed in Leishmania cell-free lysates. More than 1000 events were collected in all cases.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Two-color single molecule fluorescence coincidence of BRCA1-GFP with (A). mCherry control, (B). mCherry-cavin1, (C). mCherry-cavin2, (D). mCherry-cavin3, (E). mCherry-CAV1 coexpressed in MCF-7 cells. The green curve represents BRCA1-GFP only events, the red curve represents mCherry only events and the yellow curve represents BRCA1-GFP + Cherry events. (F). Distribution of burst brightness measured for BRCA1-GFP (blue) and GFP control (green). (G). Schematic representation of domain organization of full-length wildtype (WT) BRCA1 and the truncated (Tr) 1-300 BRCA1 constructs. Nuclear export signal-NES, BRCA1 C Terminus domain (BRCT) domain, N-N Terminus (H-J). Two-color single molecule fluorescence coincidence of 1-300 BRCA1 with (H). cavin1, (I). cavin2 and (J). cavin3 expressed in Leishmania cell-free lysates. More than 1000 events were collected in all cases.

Techniques: Fluorescence, Control, Construct

Two-color single molecule fluorescence coincidence of BRCA1-GFP with mCherry tagged (A). cavin1, (B). cavin2, (C). cavin3, (D). CAV1 and (E). mCherry control expressed in MDA-MB231 cells. The green curve represents BRCA1-GFP only events, the red curve represents mCherry only events and the yellow curve represents BRCA1-GFP + Cherry events. More than 1000 events were collected.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: Two-color single molecule fluorescence coincidence of BRCA1-GFP with mCherry tagged (A). cavin1, (B). cavin2, (C). cavin3, (D). CAV1 and (E). mCherry control expressed in MDA-MB231 cells. The green curve represents BRCA1-GFP only events, the red curve represents mCherry only events and the yellow curve represents BRCA1-GFP + Cherry events. More than 1000 events were collected.

Techniques: Fluorescence, Control

(A-E). Immunofluorescence microscopy in combination with PLA for protein-protein interactions (red dots) within single cells of stably expressing (A). MCF7/GFP, (B). MCF7/cavin1-GFP, (C). MCF7/cavin2-GFP (D). MCF7/cavin3-GFP and (E ). MCF7/CAV1-GFP using monoclonal GFP and polyclonal BRCA1 antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (F). Number of red dots/PLA signals in 40-50 cells for each MCF7/GFP expressing cell line was quantified from 3 independent experiments using a nested ANOVA Each biological replicate is color coded and the Mean ± SEM is presented as a black bar. ** p<0.05, ** p<0.01.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A-E). Immunofluorescence microscopy in combination with PLA for protein-protein interactions (red dots) within single cells of stably expressing (A). MCF7/GFP, (B). MCF7/cavin1-GFP, (C). MCF7/cavin2-GFP (D). MCF7/cavin3-GFP and (E ). MCF7/CAV1-GFP using monoclonal GFP and polyclonal BRCA1 antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (F). Number of red dots/PLA signals in 40-50 cells for each MCF7/GFP expressing cell line was quantified from 3 independent experiments using a nested ANOVA Each biological replicate is color coded and the Mean ± SEM is presented as a black bar. ** p<0.05, ** p<0.01.

Techniques: Immunofluorescence, Microscopy, Protein-Protein interactions, Stable Transfection, Expressing

(A). Immunofluorescence microscopy in combination with PLA to detect and visualize protein-protein interactions (red dots) within single cells of stably expressing MCF7/GFP and (B). MCF7/cavin3-GFP cells using monoclonal BRCA1 (MS 110) and polyclonal GFP antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (C) . Number of red dots/PLA signals in 40-50 cells for each MCF7/GFP expressing cell line quantified from 3 independent experiments using a nested ANOVA. Each biological replicate is color coded with the Mean ± SEM presented as a black bar. ** p<0.01.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Immunofluorescence microscopy in combination with PLA to detect and visualize protein-protein interactions (red dots) within single cells of stably expressing MCF7/GFP and (B). MCF7/cavin3-GFP cells using monoclonal BRCA1 (MS 110) and polyclonal GFP antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (C) . Number of red dots/PLA signals in 40-50 cells for each MCF7/GFP expressing cell line quantified from 3 independent experiments using a nested ANOVA. Each biological replicate is color coded with the Mean ± SEM presented as a black bar. ** p<0.01.

Techniques: Immunofluorescence, Microscopy, Protein-Protein interactions, Stable Transfection, Expressing

(A). Fluorescence microscopy analysis of PLA signals generated in MCF7 cells transfected with cavin3-GFP using mouse GFP and rabbit BRCA1 antibodies from , (B). GFP antibody alone, (C). BRCA1 antibody alone, (D). the absence of PLA probes or (E). primary antibody controls. Representative images are from at least two independent experiments as shown.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Fluorescence microscopy analysis of PLA signals generated in MCF7 cells transfected with cavin3-GFP using mouse GFP and rabbit BRCA1 antibodies from , (B). GFP antibody alone, (C). BRCA1 antibody alone, (D). the absence of PLA probes or (E). primary antibody controls. Representative images are from at least two independent experiments as shown.

Techniques: Fluorescence, Microscopy, Generated, Transfection

(A). Representative image of MCF7 cells stably expressing GFP alone, cavin1/GFP and cavin3/GFP fixed and stained with a BRCA1 antibody. (B). Percentage of MCF7 cells showing strictly nuclear, nuclear-cytoplasmic or cytoplasmic localization of BRCA1 was counted for 50 cells from 4-5 independent experiments as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. Each biological replicate was color-coded. NS – not significant, * p<0.05, ** p<0.01. (C) Lysates from stably expressing MCF7 cells Western blotted for GFP, BRCA1 and Tubulin as a load control. (D). MCF7/GFP and MCF7/cavin3-GFP cells, untreated (-) or treated with MG-132 for 6 h. Lysates were Western blotted with GFP, BRCA1, and Tubulin antibodies as a loading control. (E). A431 cells treated with control siRNAs (Con) or two siRNAs specific to cavin3. Lysates were Western blotted using cavin3, BRCA1, CAV1 antibodies and Tubulin as the loading control. (F). A431 cells treated with control siRNAs or two siRNAs specific to BRCA1. Lysates were Western blotted using cavin3, BRCA1, CAV1 antibodies and Tubulin as the loading control. (G). A431 cells treated with Control (Con) or siRNAs specific to cavin3, untreated or treated with MG132 for 6 hours. Lysates were Western blotted using cavin3, BRCA1, and Tubulin as a loading control. Quantitation of all blots in are provided in .

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Representative image of MCF7 cells stably expressing GFP alone, cavin1/GFP and cavin3/GFP fixed and stained with a BRCA1 antibody. (B). Percentage of MCF7 cells showing strictly nuclear, nuclear-cytoplasmic or cytoplasmic localization of BRCA1 was counted for 50 cells from 4-5 independent experiments as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. Each biological replicate was color-coded. NS – not significant, * p<0.05, ** p<0.01. (C) Lysates from stably expressing MCF7 cells Western blotted for GFP, BRCA1 and Tubulin as a load control. (D). MCF7/GFP and MCF7/cavin3-GFP cells, untreated (-) or treated with MG-132 for 6 h. Lysates were Western blotted with GFP, BRCA1, and Tubulin antibodies as a loading control. (E). A431 cells treated with control siRNAs (Con) or two siRNAs specific to cavin3. Lysates were Western blotted using cavin3, BRCA1, CAV1 antibodies and Tubulin as the loading control. (F). A431 cells treated with control siRNAs or two siRNAs specific to BRCA1. Lysates were Western blotted using cavin3, BRCA1, CAV1 antibodies and Tubulin as the loading control. (G). A431 cells treated with Control (Con) or siRNAs specific to cavin3, untreated or treated with MG132 for 6 hours. Lysates were Western blotted using cavin3, BRCA1, and Tubulin as a loading control. Quantitation of all blots in are provided in .

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Control, Quantitation Assay

(A). Relative protein expression of BRCA1 in MCF7 cells expressing GFP (white), cavin1-GFP (blue) and cavin3-GFP (orange) using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (B). BRCA1 gene expression was analysed using the Taqman Gene Expression assay as described in Materials and Methods in MCF7 cells. Results are expressed as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. NS, not significant. (C). Relative protein expression of BRCA1 in MCF7 cells expressing GFP or cavin3-GFP untreated (-) or treated with MG132 (+) using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (D). Relative protein levels of cavin3 and BRCA1 in cells treated with control (Con) siRNAs or siRNAs specific to cavin3 (oligo1 and oligo2) in A431 cells using a one-way ANOVA and Bonferroni’s multiple comparisons test from three-four independent experiments. (E). Representative Western blots of MCF7, MDA-MB231, A431 and HeLa cells were Western blotted for protein expression of cavin3, CAV1, BRCA1 and Tubulin as the loading control (F). Relative protein levels of cavin3 and BRCA1 in cells treated with control (Con) siRNAs or siRNAs specific to BRCA1 (oligo1 and oligo2) in A431 cells using a one-way ANOVA and Bonferroni’s multiple comparisons test from three-four independent experiments. For each experiment, each biological replicate was color coded. NS – not significant, * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Relative protein expression of BRCA1 in MCF7 cells expressing GFP (white), cavin1-GFP (blue) and cavin3-GFP (orange) using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (B). BRCA1 gene expression was analysed using the Taqman Gene Expression assay as described in Materials and Methods in MCF7 cells. Results are expressed as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. NS, not significant. (C). Relative protein expression of BRCA1 in MCF7 cells expressing GFP or cavin3-GFP untreated (-) or treated with MG132 (+) using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (D). Relative protein levels of cavin3 and BRCA1 in cells treated with control (Con) siRNAs or siRNAs specific to cavin3 (oligo1 and oligo2) in A431 cells using a one-way ANOVA and Bonferroni’s multiple comparisons test from three-four independent experiments. (E). Representative Western blots of MCF7, MDA-MB231, A431 and HeLa cells were Western blotted for protein expression of cavin3, CAV1, BRCA1 and Tubulin as the loading control (F). Relative protein levels of cavin3 and BRCA1 in cells treated with control (Con) siRNAs or siRNAs specific to BRCA1 (oligo1 and oligo2) in A431 cells using a one-way ANOVA and Bonferroni’s multiple comparisons test from three-four independent experiments. For each experiment, each biological replicate was color coded. NS – not significant, * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001

Techniques: Expressing, Gene Expression, Control, Western Blot

(A). Representative Western blot analysis of cavin3, BRCA1, CAV1 and Tubulin as the loading control in MDA-MB231 cells following treatment with two siRNAs targeting cavin3 oligo 1 and 2 (or control). (B) . Relative protein expression of cavin3 and BRCA1 in MDA-MB 231 cells transfected with cavin3 oligo 1 and cavin3 oligo 2 compared to control treated cells from three independent experiments using a Oneway ANOVA with Bonferroni multiple comparison tests. Each biological replicate is color coded. (C). Western blot analysis of BRCA1, cavin3 and Tubulin as the loading control in MDA-MB 231 cells following treatment with two siRNAs targeting BRCA1 oligo 1 and 2 (or control). (D) . Relative protein expression of cavin3 and BRCA1 in MDA-MB 231 cells transfected with cavin3 oligo 1 and cavin3 oligo 2 compared to control treated cells from three independent experiments using a one-way ANOVA with Bonferroni multiple comparison test. Each biological replicate is color coded. ** p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Representative Western blot analysis of cavin3, BRCA1, CAV1 and Tubulin as the loading control in MDA-MB231 cells following treatment with two siRNAs targeting cavin3 oligo 1 and 2 (or control). (B) . Relative protein expression of cavin3 and BRCA1 in MDA-MB 231 cells transfected with cavin3 oligo 1 and cavin3 oligo 2 compared to control treated cells from three independent experiments using a Oneway ANOVA with Bonferroni multiple comparison tests. Each biological replicate is color coded. (C). Western blot analysis of BRCA1, cavin3 and Tubulin as the loading control in MDA-MB 231 cells following treatment with two siRNAs targeting BRCA1 oligo 1 and 2 (or control). (D) . Relative protein expression of cavin3 and BRCA1 in MDA-MB 231 cells transfected with cavin3 oligo 1 and cavin3 oligo 2 compared to control treated cells from three independent experiments using a one-way ANOVA with Bonferroni multiple comparison test. Each biological replicate is color coded. ** p<0.01, ***p<0.001.

Techniques: Western Blot, Control, Expressing, Transfection, Comparison

Representative immunofluorescence images of Control knockdown, cavin3 knockdown and BRCA1 knockdown cells for cavin3 (green), BRCA1(red) and DAPI (nuclei). Images are representative of three independent experiments.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: Representative immunofluorescence images of Control knockdown, cavin3 knockdown and BRCA1 knockdown cells for cavin3 (green), BRCA1(red) and DAPI (nuclei). Images are representative of three independent experiments.

Techniques: Immunofluorescence, Control, Knockdown

(A). A431 cells were treated with isotonic (ISO) or hypo-osmotic (HYPO) medium and PLA was performed using cavin3 and BRCA1 (B). cavin3 and flotillin1 (C). cavin3 and PCNA and (D). cavin3 and Aurora kinase antibodies as controls for PLA. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (E). Total number of PLA signals in the cytosol and the nucleus of cells as defined by DAPI staining in 50 cells for each pair of antibodies quantified from three independent experiments using a nested ANOVA with the Mean ± SEM represented by the black bar, * p<0.05, **p<0.01.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). A431 cells were treated with isotonic (ISO) or hypo-osmotic (HYPO) medium and PLA was performed using cavin3 and BRCA1 (B). cavin3 and flotillin1 (C). cavin3 and PCNA and (D). cavin3 and Aurora kinase antibodies as controls for PLA. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (E). Total number of PLA signals in the cytosol and the nucleus of cells as defined by DAPI staining in 50 cells for each pair of antibodies quantified from three independent experiments using a nested ANOVA with the Mean ± SEM represented by the black bar, * p<0.05, **p<0.01.

Techniques: Staining

(A). Immunofluorescence microscopy in combination with PLA visualization of endogenous proteinprotein interactions (red dots) within A431 cells in (A). Untreated (Unt.) cells, (B). UV treated and a chase time of 30 min, (C) . 200 μM H 2 0 2 (H 2 0 2 ) for 30 min and (D). Hypo-osmotic treatment (HYPO) for 10 min, top panel–BRCA1 and cavin3 and bottom panel-cavin1 and cavin3. (E). PLA signals/cell for cavin3-BRCA1 association in 50 cells/biological replicate with three independent experiments. (F). PLA time course analysis after UV treatment and a chase time up to 360 min in 50 cells/biological replicate with three independent experiments. (G). PLA signals/cell for cavin1-cavin3 association in 50 cells/biological replicate with three independent experiments. All data was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color coded with the Mean ± SEM presented as a black bar. NS-not significant, * p<0.05, ** p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Immunofluorescence microscopy in combination with PLA visualization of endogenous proteinprotein interactions (red dots) within A431 cells in (A). Untreated (Unt.) cells, (B). UV treated and a chase time of 30 min, (C) . 200 μM H 2 0 2 (H 2 0 2 ) for 30 min and (D). Hypo-osmotic treatment (HYPO) for 10 min, top panel–BRCA1 and cavin3 and bottom panel-cavin1 and cavin3. (E). PLA signals/cell for cavin3-BRCA1 association in 50 cells/biological replicate with three independent experiments. (F). PLA time course analysis after UV treatment and a chase time up to 360 min in 50 cells/biological replicate with three independent experiments. (G). PLA signals/cell for cavin1-cavin3 association in 50 cells/biological replicate with three independent experiments. All data was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color coded with the Mean ± SEM presented as a black bar. NS-not significant, * p<0.05, ** p<0.01, ***p<0.001.

Techniques: Immunofluorescence, Microscopy

(A). Immunofluorescence microscopy in combination with PLA visualization of endogenous protein–protein interactions (red dots) within MDA-MB231 cells in untreated (Unt.) cells, (B). UV treatment (2 min) and a 30 min chase time, (C). 200 μM H 2 0 2 for 30 min and (D). Hypo-osmotic treatment (90% H 2 0 in DMEM) for 10 min. (E). Number of red dots/PLA signals in 40-50 cells for cavin-BRCA1 was quantified from 3 independent experiments and is presented as Mean ± SEM using a nested ANOVA. Each biological replicate is color coded with the mean presented as a black bar. (F). Number of red dots/PLA signals in 40-50 cells for cavin3-cavin1 was quantified from 3 independent experiments and is presented as Mean ± SEM using a nested ANOVA. Each biological replicate is color coded with the mean presented as a black bar. * p<0.05, ** p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Immunofluorescence microscopy in combination with PLA visualization of endogenous protein–protein interactions (red dots) within MDA-MB231 cells in untreated (Unt.) cells, (B). UV treatment (2 min) and a 30 min chase time, (C). 200 μM H 2 0 2 for 30 min and (D). Hypo-osmotic treatment (90% H 2 0 in DMEM) for 10 min. (E). Number of red dots/PLA signals in 40-50 cells for cavin-BRCA1 was quantified from 3 independent experiments and is presented as Mean ± SEM using a nested ANOVA. Each biological replicate is color coded with the mean presented as a black bar. (F). Number of red dots/PLA signals in 40-50 cells for cavin3-cavin1 was quantified from 3 independent experiments and is presented as Mean ± SEM using a nested ANOVA. Each biological replicate is color coded with the mean presented as a black bar. * p<0.05, ** p<0.01, ***p<0.001.

Techniques: Immunofluorescence, Microscopy, Protein-Protein interactions

A431 cells treated with control (Con) or siRNAs specific to cavin3 or BRCA1 (oligo1 and 2). Cells were left untreated or subjected to UV treatment. Cells were subjected to immunofluorescence microscopy in combination with PLA (red dots) using monoclonal BRCA1 and polyclonal cavin3 antibodies. The number of red dots/PLA signals in 40-50 cells was quantified from 3 independent experiments using a nested ANOVA. Each biological replicate is color coded with the Mean ± SEM presented as a black bar. *p<0.05, **p<0.01.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: A431 cells treated with control (Con) or siRNAs specific to cavin3 or BRCA1 (oligo1 and 2). Cells were left untreated or subjected to UV treatment. Cells were subjected to immunofluorescence microscopy in combination with PLA (red dots) using monoclonal BRCA1 and polyclonal cavin3 antibodies. The number of red dots/PLA signals in 40-50 cells was quantified from 3 independent experiments using a nested ANOVA. Each biological replicate is color coded with the Mean ± SEM presented as a black bar. *p<0.05, **p<0.01.

Techniques: Control, Immunofluorescence, Microscopy

(A) LDH release of MCF7/GFP, cavin3-GFP and cavin1-GFP cells subjected to UV treatment and a 6 h chase. LDH release is expressed as a percentage to control GFP cells from six independent experiments presented as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. (B). Annexin V positive cells after UV treatment and a 6 h recovery time in MCF7 cells presented as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (C) 7-AAD positive cells after UV treatment and a 24 h recovery time in MCF7 cells presented as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (D). A431 cells and (E). MDA-MB231 cells were transfected with GFP, cavin3-GFP or BRCA1-GFP. Results are the relative percentage of LDH release to GFP as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from at least 3 independent experiments. (F) A431 cells and (G). MDA-MB231 cells were treated with control, cavin3 or BRCA1 specific siRNAs. Cavin3-depleted A431 and MDA-MB231 cells were transfected with BRCA1-GFP for 24 hours. BRCA1 depleted A431 and MDA-MB231 cells were transfected with cavin3-GFP for 24 hours. All cells were UV treated and LDH release was measured and calculated relative to control siRNA UV treated cells. The results represent independent experiments as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. Each biological replicate is color coded. NS – not significant, * p<0.05, ** p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A) LDH release of MCF7/GFP, cavin3-GFP and cavin1-GFP cells subjected to UV treatment and a 6 h chase. LDH release is expressed as a percentage to control GFP cells from six independent experiments presented as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. (B). Annexin V positive cells after UV treatment and a 6 h recovery time in MCF7 cells presented as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (C) 7-AAD positive cells after UV treatment and a 24 h recovery time in MCF7 cells presented as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (D). A431 cells and (E). MDA-MB231 cells were transfected with GFP, cavin3-GFP or BRCA1-GFP. Results are the relative percentage of LDH release to GFP as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from at least 3 independent experiments. (F) A431 cells and (G). MDA-MB231 cells were treated with control, cavin3 or BRCA1 specific siRNAs. Cavin3-depleted A431 and MDA-MB231 cells were transfected with BRCA1-GFP for 24 hours. BRCA1 depleted A431 and MDA-MB231 cells were transfected with cavin3-GFP for 24 hours. All cells were UV treated and LDH release was measured and calculated relative to control siRNA UV treated cells. The results represent independent experiments as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. Each biological replicate is color coded. NS – not significant, * p<0.05, ** p<0.01, ***p<0.001.

Techniques: Control, Transfection

(A). Equal numbers of A431 cells treated with control siRNA, or cavin3 specific siRNAs were subjected to UV treatment and a recovery time of 6 hours. (B). Equal numbers of A431 cells treated with control siRNAs or BRCA1 specific siRNAs were subjected to UV treatment (2 min) and a recovery time of 6 hours. (C). Equal numbers of MDA-MB231 cells treated with control siRNAs or cavin3 specific siRNAs were subjected to UV treatment and a recovery time of 6 hours. (D). Equal numbers of MDA-MB231 cells treated with control siRNAs or BRCA1 specific siRNAs were subjected to UV treatment and a recovery time of 6 hours. LDH release was measured from the cell supernatant and was calculated relative to control siRNA UV treated cells as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. (E). MCF7 cells depleted of BRCA1 (-cavin3, -BRCA1), depleted of BRCA1 and transfected with cavin3-GFP (-BRCA1, + cavin3), left untreated (-cavin3, + BRCA1) or transfected with cavin3-GFP (+ BRCA1, + cavin3). All cells were subjected to UV treatment and a 6 hour recovery time. LDH release was measured from the cell supernatant and was calculated relative to control MCF7 cells lacking both BRCA1 and cavin3 as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. Each biological replicate is color coded. NS – not significant, * p<0.05, ***p<0.001, **** p<0.0001.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A). Equal numbers of A431 cells treated with control siRNA, or cavin3 specific siRNAs were subjected to UV treatment and a recovery time of 6 hours. (B). Equal numbers of A431 cells treated with control siRNAs or BRCA1 specific siRNAs were subjected to UV treatment (2 min) and a recovery time of 6 hours. (C). Equal numbers of MDA-MB231 cells treated with control siRNAs or cavin3 specific siRNAs were subjected to UV treatment and a recovery time of 6 hours. (D). Equal numbers of MDA-MB231 cells treated with control siRNAs or BRCA1 specific siRNAs were subjected to UV treatment and a recovery time of 6 hours. LDH release was measured from the cell supernatant and was calculated relative to control siRNA UV treated cells as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. (E). MCF7 cells depleted of BRCA1 (-cavin3, -BRCA1), depleted of BRCA1 and transfected with cavin3-GFP (-BRCA1, + cavin3), left untreated (-cavin3, + BRCA1) or transfected with cavin3-GFP (+ BRCA1, + cavin3). All cells were subjected to UV treatment and a 6 hour recovery time. LDH release was measured from the cell supernatant and was calculated relative to control MCF7 cells lacking both BRCA1 and cavin3 as Mean ± SD using a one-way ANOVA and Bonferroni’s multiple comparisons test. Each biological replicate is color coded. NS – not significant, * p<0.05, ***p<0.001, **** p<0.0001.

Techniques: Control, Transfection

(A ). Representative Western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. (B). Protein components of the BRCA1 A-complex. Blue colored circles; proteins downregulated in the LFQ proteomics and yellow colored circles; proteins not detected in the LFQ proteomics of cavin3 KO cells. (C). Representative Western blot analysis of cavin3, BRCA1, γH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1 and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hour chase. Quantitation of protein levels from three independent experiments is presented in . (D). Representative immunofluorescence images of BRCA1 foci after UV treatment in WT HeLa cells. (E). Percentage of cells with more than five BRCA1 foci, Rap80 foci and γH2AX foci in WT and cavin3 KO cells following UV treatment and a 30 min chase. The results are presented as Mean ± SD using a oneway ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (F). WT and cavin3 KO cells were treated with a PARP inhibitor (PARPi) 50 μ M AZD 2461. Presto blue reagent was added to plates immediately and were read at 570 and 600 nm at 6 hours and 24 hours. The % reduction prestoblue calculated from eight wells/replicate experiment is presented as the Mean ± SEM using a nested ANOVA from three independent experiments. Each biological replicate is color coded. * p<0.05, ** p<0.01.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: (A ). Representative Western blot analysis of WT and cavin3 KO cells UV time course for cavin3, BRCA1, CAV1, Rad51, and Tubulin. (B). Protein components of the BRCA1 A-complex. Blue colored circles; proteins downregulated in the LFQ proteomics and yellow colored circles; proteins not detected in the LFQ proteomics of cavin3 KO cells. (C). Representative Western blot analysis of cavin3, BRCA1, γH2AX, UIM1C/Rap80, BARD1, Rad51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV, PKM, PGK1 and Actin in WT and cavin3 KO HeLa cells untreated (-) or UV treated (UV) followed by a 4 hour chase. Quantitation of protein levels from three independent experiments is presented in . (D). Representative immunofluorescence images of BRCA1 foci after UV treatment in WT HeLa cells. (E). Percentage of cells with more than five BRCA1 foci, Rap80 foci and γH2AX foci in WT and cavin3 KO cells following UV treatment and a 30 min chase. The results are presented as Mean ± SD using a oneway ANOVA and Bonferroni’s multiple comparisons test from three independent experiments. (F). WT and cavin3 KO cells were treated with a PARP inhibitor (PARPi) 50 μ M AZD 2461. Presto blue reagent was added to plates immediately and were read at 570 and 600 nm at 6 hours and 24 hours. The % reduction prestoblue calculated from eight wells/replicate experiment is presented as the Mean ± SEM using a nested ANOVA from three independent experiments. Each biological replicate is color coded. * p<0.05, ** p<0.01.

Techniques: Western Blot, Quantitation Assay, Immunofluorescence

Densitometry analysis was performed of the protein levels of cavin3, BRCA1, P139 γH2AX, RAP80, BARD1, RAD51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV1, PKM, PGK1 and Actin in and in WT and cavin3 KO cells subjected to UV treatment and a 4 hour chase from two-three independent experiments presented as Mean ± SD using a one way ANOVA and Bonferroni’s multiple comparisons test. NS, not significant, * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.

Journal: bioRxiv

Article Title: Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response

doi: 10.1101/2020.07.26.222158

Figure Lengend Snippet: Densitometry analysis was performed of the protein levels of cavin3, BRCA1, P139 γH2AX, RAP80, BARD1, RAD51, MDC1, RNF168, BRCC36, Merit40, BRCA2, CAV1, PKM, PGK1 and Actin in and in WT and cavin3 KO cells subjected to UV treatment and a 4 hour chase from two-three independent experiments presented as Mean ± SD using a one way ANOVA and Bonferroni’s multiple comparisons test. NS, not significant, * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.

Techniques:

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