Complete mini protease and phosphatase inhibitor cocktails

Cells were lysed with RIPA buffer (Sigma) supplemented with Complete Mini protease and phosphatase inhibitor cocktails (Roche). 5 μg of proteins were loaded on NuPAGE 4–12% Bis-Tris gel and transferred on Nitrocellulose membranes with iBlot system (Life Technologies).
After blocking in 5% milk-TBST, membranes were incubated overnight with primary antibodies anti-FLAG (M2, Sigma RRID: AB_259529) and anti-GAPDH (sc-25778, Santa Cruz Biotechnology RRID : AB_10167668).
After 1 hour incubation in the appropriate HRP conjugated secondary antibodies (Merck Millipore), detection was performed with ECL Star (Euroclone) with Azure Biosystem C400 camera. Densitometric quantification was performed with FIJI (RRID : SCR_002285) (26 (link)).

Cells were harvested in EBC lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40), supplemented with Complete Mini Protease and Phosphatase Inhibitor Cocktails (Roche, Indianapolis, IN, USA). Cells were lysed and 30-80 μg protein subjected to SDS-PAGE followed by transfer onto an Immobilon-FL PVDF membrane (Millipore, Billerica, MA, USA) and incubation with the indicated antibodies. Detection was carried out with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) with IR dye-tagged secondary antibodies (LI-COR Biosciences). The following antibodies were utilized: mouse anti-YAP, goat anti-NF2, mouse anti-AMOT, goat anti-AMOTL1, goat anti-AMOTL2 (Santa Cruz, Dallas, TX, USA), mouse anti-FlagM2 (Sigma, Saint Louis, MO, USA), rabbit anti-LATS1, rabbit anti-LATS2, rabbit anti-p-YAP (Cell Signaling, Danvers, MA, USA), TNKS1/2 (Santa Cruz, Dallas, TX, USA), mouse anti-TEAD4, mouse anti-RAS (Thermo Scientific, Waltham, MA, USA), mouse anti-α-Tubulin, mouse anti-β-actin (Sigma, Saint Louis, MO, USA).

Cells were harvested in EBC lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40), supplemented with Complete Mini Protease and Phosphatase Inhibitor Cocktails (Roche, Indianapolis, IN, USA). Cells were lysed and 30–80 µg protein subjected to SDS-PAGE followed by transfer onto an Immobilon-FL PVDF membrane (Millipore, Billerica, MA, USA) and incubation with the indicated antibodies. Detection was carried out with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) with IR dye-tagged secondary antibodies (Alexa Fluor 750 goat anti rabbit IgG; Alexa Fluor 750 goat anti mouse IgG, Alexa Fluor 680 goat anti rabbit IgG, Alexa Fluor 680 goat anti mouse IgG from Invitrogen, 1:10,000 dilution). The following antibodies were utilized: p53 (1801; Mount Sinai School of Medicine Hybridoma Center, New York, NY, USA, 1:1000 dilution), YAP, CTGF, MYC, MLC2, RhoA, pan-Rac, Cdc42, ROCK1, ROCK2, GAPDH (Santa Cruz, Dallas, TX, USA, 1:1000 dilution), p-YAP, p-Cofilin, p-MLC2, Cofilin, pPAK1/2, pPAK4/5, PAK2, PAK4, (Cell Signaling, Danvers, MA, USA, 1:1000 dilution), TEAD4, (Thermo Scientific, Waltham, MA, USA, 1:1000 dilution), mouse anti-α-tubulin (1:10,000 dilution), mouse anti-β-actin (Sigma, Saint Louis, MO, USA, 1:10,000 dilution).