Spheroid Lysis and Protein Analysis

Spheroids were lysed in lysis buffer (0.5% TritonX-100, 150 mM NaCl, 20 mM Tris-HCl pH7.5). The lysates were sheared with a 21-gauge needle, incubated on ice for 30 minutes and clarified by centrifugation at 20,817 × g for 15 minutes at 4°C. The extracted proteins were separated by SDS-PAGE and transferred to immobilon transfer membrane (Millipore) for Western blotting analyses. The primary antibodies were anti-YAP1 pAb (#4912 Cell Signaling, 1:500), anti-Fibronectin pAb (F3648, Sigma Aldrich, 1:1000), anti-ARHGAP18 pAb (1:10000)^{17 (link),21 (link)}, anti-MYH9 pAb (#3403 Cell signaling, 1:1000), anti-Phospho Ser1943-MYH9 pAb (#5026 Cell Signaling, 1:1000), anti-MYH10 mAb (#8824 Cell Signaling, 1:1000), anti-Phospho-Ser19 MLC2 (#3675, Cell signaling, 1:100), and anti-GAPDH mAb (sc32233, Santa Cruz, 1:5000).

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Porazinski S., Wang H., Asaoka Y., Behrndt M., Miyamoto T., Morita H., Hata S., Sasaki T., Krens S.F., Osada Y., Asaka S., Momoi A., Linton S., Miesfeld J.B., Link B.A., Senga T., Shimizu N., Nagase H., Matsuura S., Bagby S., Kondoh H., Nishina H., Heisenberg C.P, & Furutani-Seiki M. (2015). YAP is essential for tissue tension to ensure vertebrate 3D body shape. Nature, 521(7551), 217-221.

Publication 2015

immobilon transfer membrane anti-YAP1 pAb anti-Fibronectin pAb anti-ARHGAP18 pAb anti-MYH9 pAb anti-Phospho Ser1943-MYH9 pAb anti-MYH10 mAb anti-Phospho-Ser19 MLC2 anti-GAPDH mAb

Antibodies Buffer Centrifugation Fibronectin Gapdh Immobilon Membrane Nacl Needle Proteins Sds page Tris Western blotting analyses Yap1

Corresponding Organization :

Other organizations : University of Bath, Tokyo Medical and Dental University, Institute of Science and Technology Austria, Hiroshima University, Keio University, Japan Science and Technology Agency, Medical College of Wisconsin, Nagoya University, University of Oxford, Osaka University, Kyoto Sangyo University

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Variable analysis

independent variables

Lysis buffer (0.5% TritonX-100, 150 mM NaCl, 20 mM Tris-HCl pH7.5)
Shearing with a 21-gauge needle
Incubation on ice for 30 minutes
Centrifugation at 20,817 × g for 15 minutes at 4°C

dependent variables

Extracted proteins
Protein expression levels of YAP1, Fibronectin, ARHGAP18, MYH9, Phospho Ser1943-MYH9, MYH10, Phospho-Ser19 MLC2, and GAPDH

control variables

Spheroids

controls

Positive control: Not specified
Negative control: Not specified

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