Hpa007415, by Merck Group - Product details

1

Immunofluorescence Staining of MEFs

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MEFs were plated on 8-well Nunc Lab-Tek II chamber slides (Sigma-Aldrich) and fixed with 4% paraformaldehyde for 15 min at 4 °C. After several washes with PBS, samples were blocked with 5% goat (or donkey) serum in 0.5% PBST for 30 min at RT. Cells were incubated with the indicated primary antibodies at 4 °C overnight. Bound primary antibodies were detected by incubating with secondary antibodies for 90 min at RT. The following primary antibodies were used for the cell staining: anti-YAP (rabbit monoclonal, 14074, Cell signaling); anti-TAZ (rabbit polyclonal, HPA007415, Sigma-Aldrich); and Alexa Fluor 488-conjugated anti-phalloidin (A12379, Thermo Fisher) antibodies. Alexa Fluor 594-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. Nuclei were stained with DAPI (Invitrogen) and slides were mounted and imaged as described above.

Hong S.P., Yang M.J., Cho H., Park I., Bae H., Choe K., Suh S.H., Adams R.H., Alitalo K., Lim D, & Koh G.Y. (2020). Distinct fibroblast subsets regulate lacteal integrity through YAP/TAZ-induced VEGF-C in intestinal villi. Nature Communications, 11, 4102.

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2

Nuclear Protein Extraction and Immunoprecipitation

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Cells were rinsed twice with ice-cold HBSS and incubated with ice-cold hypotonic buffer (2 x 1min, 20mM HEPES, 20% Glycerol, 10mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA, 0.1% NP40, freshly supplemented with 1 mM DTT, and protease and phosphatase inhibitor cocktails). Nuclei were then harvested by scraping in hypertonic buffer (hypotonic buffer + 500mM NaCl, 400 µl/60 cm²) and disrupted by sonication in a water-bath sonicator. Nuclear lysates were cleared by centrifugation and quantified by Bradford. For immunoprecipitation, extracts were diluted to 140mM NaCl and incubated 4h at 4°C with magnetic beads (Dynabeads Protein A or G, Invitrogen) preloaded with specific primary antibodies. Immunocomplexes were then washed in binding buffer four times; finally, beads were resuspended in SDS sample buffer.
Antibodies used for immunoprecipitation: anti-BRD4 (E2A7X, CST); anti-YAP1 (13584-1-AP, Proteintech); anti-WWTR1 (HPA007415, Sigma); anti-FLAG (clone M2, A8592, Sigma); normal rabbit IgG (I5006, Sigma).

Zanconato F., Battilana G., Forcato M., Filippi L., Azzolin L., Manfrin A., Quaranta E., Di Biagio D., Sigismondo G., Guzzardo V., Lejeune P., Haendler B., Krijgsveld J., Fassan M., Bicciato S., Cordenonsi M, & Piccolo S. (2018). Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4. Nature medicine, 24(10), 1599-1610.

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3

In Situ Proximity Ligation Assay for BRD4 Interactome

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HEK293T cells were seeded on fibronectin-coated glass chamber slides and transfected with pFlag-CMV2-BRD4, pCS2-HA-BRD4 or empty pCS2+ as negative control. After 24 hours, cells were fixed in 4% PFA for 10 min at RT. In situ PLA was performed with DuoLink In Situ Reagents (Sigma) according to manufacturer’s instructions. Primary antibodies used in the PLA are: mouse anti-HA (F-7, sc-7392, SantaCruz), mouse anti-TEF1 (610923; BD Biosciences), rabbit anti-FLAG (F-7425; Sigma), rabbit anti-YAP1 (EP1674Y, abcam), rabbit anti-WWTR1 (HPA007415, Sigma). Images were acquired with a Leica TCS SP5 confocal microscope equipped with a CCD camera; for each field, a Z-stack was acquired; images were processed using Volocity software (PerkinElmer). We verified that the fraction of nuclei with positive PLA signal corresponded to the fraction of transfected cells (determined by immunofluorescence for FLAG or HA).

Zanconato F., Battilana G., Forcato M., Filippi L., Azzolin L., Manfrin A., Quaranta E., Di Biagio D., Sigismondo G., Guzzardo V., Lejeune P., Haendler B., Krijgsveld J., Fassan M., Bicciato S., Cordenonsi M, & Piccolo S. (2018). Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4. Nature medicine, 24(10), 1599-1610.

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4

Immunohistochemical Analysis of TAZ in GBM

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Archival frozen GBM specimens were collected at the Azienda Ospedaliera,
Padua, Italy. For IHC, 4 μm thick sections were obtained from tumor
samples. IHC was performed with rabbit polyclonal anti-TAZ (Sigma,
HPA007415;1:50 diluted) as previously described^{73 (link)}. For mouse tissues, IHC was performed using a
fully automated system (Bond-maX; Leica).
Slide images were captured using the D-Sight-F system for digital
pathology (Menarini Diagnostics) and the percentage of TAZ-positive nuclei was
determined using the Nuclei Analysis module of the D-Sight Viewer software.

Castellan M., Guarnieri A., Fujimura A., Zanconato F., Battilana G., Panciera T., Sladitschek H.L., Contessotto P., Citron A., Grilli A., Romano O., Bicciato S., Fassan M., Porcù E., Rosato A., Cordenonsi M, & Piccolo S. (2020). Single-cell analyses reveal YAP/TAZ as regulators of stemness and cell plasticity in Glioblastoma. Nature cancer, 2(2), 174-188.

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5

Immunohistochemical Profiling of Glioblastoma Tissue

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Four μm-thick sections of paraffin-embedded samples of glioblastoma tissue from patients treated at the Instituto de Investigación Sanitaria La Paz (IdiPaz, Madrid, Spain) were arrayed in a collection of three tissue microarray slides. Taken together, these slides encompassed 26 good tumor cores. Sections were deparaffinized and rehydrated in water, and antigen retrieval was carried out by incubation in 1 mM EDTA, 0.05% Tween 20, pH 8.0 at 50 °C for 45 min. Endogenous peroxidase and nonspecific antibody reactivity was blocked with peroxidase blocking reagent (Dako) at room temperature for 15 min. The sections were then incubated for 60–90 min at 4 °C with the corresponding peroxidase conjugated primary antibodies for NRF2 (PA1-38312, Thermo Fisher Scientific), TAZ (HPA007415, Sigma-Aldrich), NQO1 (ab34173, Abcam), ATRX (DIA-AX1, Dianova), IDH1 (DIA-H09, Dianova), ki67 (M7240, Dako) and developed with 3,3′-diaminobenzidine (DAB). Negative controls with goat serum replacing the primary antibody were used. The slides were mounted with DPX (VWR International). Detection was performed with the Envision Plus Detection System (Dako). All tumors were negative for IDH1 and ATRX mutations, therefore confirming that according to histological classification they were GBs. Densitometric quantification was done using macros of the ImageJ software.

Escoll M., Lastra D., Pajares M., Robledinos-Antón N., Rojo A.I., Fernández-Ginés R., Mendiola M., Martínez-Marín V., Esteban I., López-Larrubia P., Gargini R, & Cuadrado A. (2020). Transcription factor NRF2 uses the Hippo pathway effector TAZ to induce tumorigenesis in glioblastomas. Redox Biology, 30, 101425.

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6

Antibody Validation for Western Blot and Immunofluorescence

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The antibodies used for western blot and immunofluorescence were: anti-p53 (1:1000; DO-1, Santa Cruz Biotechnology), anti-Actin (1:5000; C11, Sigma), anti-GAPDH (1:5000; MAB374, Millipore), anti-SREBP1 (2A4) (1:500; sc13551, Santa Cruz Biotechnology), anti-SREBP2 (1:500; 557037, BD Bioscience), anti-SCD-1 (1:1000; ab19862, Abcam), Anti-Vinculin (1:5000; V4505 Sigma), anti-Hsp90 (1:1000; sc13119, Santa Cruz Biotechnology), anti-HDAC6 (H-300) (1:1000; sc-11420, Santa Cruz Biotecnology), Acetylated-Lysine Antibody (1:1000; 9441, Cell Signaling), anti-α-Tubulin (1:5000, T5168, Sigma), anti-acetylated-tubulin (1:1000; T6793, Sigma), anti GFP (1:1000; home-made), anti-MDM2 (SMP14; SC-965, Santa Cruz Biotechnology), anti-MLC2 (1:1000; 3672S, Cell Signaling), anti-pMLC2 (phospho Ser19) (1:1000; 3675S; Cell signalling), anti-FAK (C-20) (1:1000; sc-558, Santa Cruz Biotechnology), anti-pFAK (phospho Y397) (1:1000; ab81298, Abcam), anti-YAP (1:1000; sc-15407; Santa Cruz Biotechnology), anti-TAZ (1:1000; HPA007415, Sigma), Anti-PSMA2 (1:1000; sc-54671; Santa Cruz). Phalloidin is A12379 (Alexa Fluor), Anti-BrdU antibody (RPN202) is GE Healthcare. anti- cleaved PARP p85 fragment pAb is from Promega (G7341).

Ingallina E., Sorrentino G., Bertolio R., Lisek K., Zannini A., Azzolin L., Severino L.U., Scaini D., Mano M., Mantovani F., Rosato A., Bicciato S., Piccolo S, & Del Sal G. (2017). Mechanical cues control mutant p53 stability through a Mevalonate/RhoA axis. Nature cell biology, 20(1), 28-35.

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7

Comprehensive Antibody Validation for WB and IF

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Antibodies used for WB and immunofluorescence: anti-YAP is sc101199 (1:1,000, Santa Cruz Biotechnology), anti- GR is BK3660S (1:1,000, Cell Signaling), anti-actin is C11 (1:2,000, Sigma), anti-ANKRD1 is 11427-1-AP (1:1,000, Proteintech (DBA), anti-pYAP (Ser127) is 4911S (1:1,000, Cell Signaling), anti-WWTR1 is HPA007415 (1:1000, Sigma), phalloidin is A12379 (1:250, Alexa Fluor), anti-PARP-85 is TB273 (1:500, Promega), anti-vinculin is V4505 (1:4,000, Sigma), anti-LATS1 is ab70562 (1:500, Abcam), phospho-LATS1 (Thr1079) is BK8654S (1:500, Cell Signaling), anti-FAK (C-20) is sc-558 (1:1,000, Santa Cruz), anti-FAK (phospho Y397) is ab81298 (1:1,000, Abcam), anti-Src is BK2110S and anti-Phospho-Src (Tyr416) is BK2101S from Cell Signaling (1:1,000), anti-FN1 is GTX112794 (1:1,000, GeneTex), anti-Slug is C19G7 (1:500, Cell Signaling), anti-Mst1 is 3682S (1:500, Cell Signaling) and anti-phospho-Mst1/2 (T183/T180) is 3681S (1:500, Cell Signaling).

Sorrentino G., Ruggeri N., Zannini A., Ingallina E., Bertolio R., Marotta C., Neri C., Cappuzzello E., Forcato M., Rosato A., Mano M., Bicciato S, & Del Sal G. (2017). Glucocorticoid receptor signalling activates YAP in breast cancer. Nature Communications, 8, 14073.

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8

Immunohistochemical Analysis of TAZ in GBM

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Archival frozen GBM specimens were collected at the Azienda Ospedaliera,
Padua, Italy. For IHC, 4 μm thick sections were obtained from tumor
samples. IHC was performed with rabbit polyclonal anti-TAZ (Sigma,
HPA007415;1:50 diluted) as previously described^{73 (link)}. For mouse tissues, IHC was performed using a
fully automated system (Bond-maX; Leica).
Slide images were captured using the D-Sight-F system for digital
pathology (Menarini Diagnostics) and the percentage of TAZ-positive nuclei was
determined using the Nuclei Analysis module of the D-Sight Viewer software.

Castellan M., Guarnieri A., Fujimura A., Zanconato F., Battilana G., Panciera T., Sladitschek H.L., Contessotto P., Citron A., Grilli A., Romano O., Bicciato S., Fassan M., Porcù E., Rosato A., Cordenonsi M, & Piccolo S. (2020). Single-cell analyses reveal YAP/TAZ as regulators of stemness and cell plasticity in Glioblastoma. Nature cancer, 2(2), 174-188.

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9

Immunochemistry Staining of Cells

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For immunochemistry staining, cells were either fixed directly on ERISM substrates or coverslips. Fixation was achieved with 4% paraformaldehyde (Alfa Aesar) in PBS at room temperature for 20 min. Cells were washed twice with 0.05% Tween-20 in PBS (Alfa Aesar), permeabilized with 0.1% Triton X-100 (Alfa Aesar) in PBS for 3 min, washed twice again, and blocked for 30 min with 1% BSA (Carl Roth) in PBS. Fixed cells on ERISM chips were simultaneously incubated with primary antibodies for vinculin (Merck Millipore, 90227, 1:250) and TAZ (Sigma–Aldrich, HPA007415, 1:100) in blocking solution for 60 min at RT, washed three times, incubated with the secondary antibodies (Sigma–Aldrich, F0257, 1:32; Jackson ImmunoResearch, 711-175-152, 1:400) and TRITC-conjugated phalloidin (Merck Millipore, 90228, 1:500) in blocking solution for 45 min at RT, washed three times, and stained with DAPI (Merck Millipore, 90229, 1:1,000 in blocking solution for 3 min at RT). Fixed cells on coverslips were incubated with a primary antibody for neuroD1 (Santa Cruz Biotechnology, sc-1084, 1:50 in blocking solution, overnight at 4°C), washed, incubated with a secondary antibody (Jackson ImmunoResearch, 205-482-176, 1:1,000 in PBS for 30 min at RT) and washed again. Antifade Mountant with DAPI (Invitrogen; P36935) was used to visualize cell nuclei.

Kronenberg N.M., Tilston-Lunel A., Thompson F.E., Chen D., Yu W., Dholakia K., Gather M.C, & Gunn-Moore F.J. (2020). Willin/FRMD6 Influences Mechanical Phenotype and Neuronal Differentiation in Mammalian Cells by Regulating ERK1/2 Activity. Frontiers in Cellular Neuroscience, 14, 552213.

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10

Nuclear Protein Extraction and Immunoprecipitation

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Cells were rinsed twice with ice-cold HBSS and incubated with ice-cold hypotonic buffer (2 x 1min, 20mM HEPES, 20% Glycerol, 10mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA, 0.1% NP40, freshly supplemented with 1 mM DTT, and protease and phosphatase inhibitor cocktails). Nuclei were then harvested by scraping in hypertonic buffer (hypotonic buffer + 500mM NaCl, 400 µl/60 cm²) and disrupted by sonication in a water-bath sonicator. Nuclear lysates were cleared by centrifugation and quantified by Bradford. For immunoprecipitation, extracts were diluted to 140mM NaCl and incubated 4h at 4°C with magnetic beads (Dynabeads Protein A or G, Invitrogen) preloaded with specific primary antibodies. Immunocomplexes were then washed in binding buffer four times; finally, beads were resuspended in SDS sample buffer.
Antibodies used for immunoprecipitation: anti-BRD4 (E2A7X, CST); anti-YAP1 (13584-1-AP, Proteintech); anti-WWTR1 (HPA007415, Sigma); anti-FLAG (clone M2, A8592, Sigma); normal rabbit IgG (I5006, Sigma).

Zanconato F., Battilana G., Forcato M., Filippi L., Azzolin L., Manfrin A., Quaranta E., Di Biagio D., Sigismondo G., Guzzardo V., Lejeune P., Haendler B., Krijgsveld J., Fassan M., Bicciato S., Cordenonsi M, & Piccolo S. (2018). Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4. Nature medicine, 24(10), 1599-1610.

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Hpa007415

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11 protocols using hpa007415

Immunofluorescence Staining of MEFs

Nuclear Protein Extraction and Immunoprecipitation

In Situ Proximity Ligation Assay for BRD4 Interactome

Immunohistochemical Analysis of TAZ in GBM

Immunohistochemical Profiling of Glioblastoma Tissue

Antibody Validation for Western Blot and Immunofluorescence

Comprehensive Antibody Validation for WB and IF

Immunohistochemical Analysis of TAZ in GBM

Immunochemistry Staining of Cells

Nuclear Protein Extraction and Immunoprecipitation

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