Inducible Expression of YAP Constructs

Doxycycline hyclate, latrunculin A, D-luciferin, DAPT and DBZ were from Sigma; fibronectin was from Santa Cruz Biotechnology; cytochalasin D was from Calbiochem; CPRG was from Roche. For the inducible expression of YAP constructs, cDNA for human YAP1 5SA (LATS-mutant sites)21 (link) and human YAP1 5SA/S94A (TEAD-binding mutant)34 (link) were made insensitive to YAP siRNA #1 and subcloned, together with an HA-tag, in FUW-tetO-MCS, obtained by substituting the Oct4 sequence in FUW-tetO-hOct4 (Addgene #20726)65 (link) with a new multiple cloning site (MCS). This generated FUW-tetO-HA-YAP5SA and FUW-tetO-HA-YAP5SA/S94A were used throughout this study. FUW-tetO-MCS (empty vector) or FUW-tetO-RFP plasmids, obtained by subcloning the RFP coding sequence from the pTomo vector (Addgene #26291), were used as controls. FUdeltaGW-rtTA was from Addgene (#19780)66 (link). FUW-tetO-N1ICD-Myc and FUW-tetO-GFP-DNMAML1 were obtained by subloning in the FUW-tetO-MCS the corresponding coding sequences, respectively, from pcDNA3 N1ICD-Myc67 (link) and MigRI-DNMAML1-GFP38 (link). All constructs were confirmed by sequencing. The 8xGTIIC-lux (Addgene #34615) was previously described22 (link). The 3D.A-Lux was gently provided by Guido Posern68 (link). Primers for RT–PCR are listed in Supplementary Data 3.

Free full text: Click here

Totaro A., Castellan M., Battilana G., Zanconato F., Azzolin L., Giulitti S., Cordenonsi M, & Piccolo S. (2017). YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate. Nature Communications, 8, 15206.

Publication 2017

Doxycycline hyclate latrunculin A D-luciferin fibronectin FUW-tetO-hOct4 FUdeltaGW-rtTA 8xGTIIC-lux

Cdna Coding sequence Coding sequences Cprg Cytochalasin d Dapt Doxycycline hyclate Fibronectin Human Latrunculin a Lats Luciferin Oct4 Plasmids Primers Rt pcr Sirna Vector Yap1

Corresponding Organization :

Other organizations : University of Padua

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

Doxycycline hyclate
Latrunculin A
D-luciferin
Human YAP1 5SA (LATS-mutant sites)
Human YAP1 5SA/S94A (TEAD-binding mutant)
FUW-tetO-HA-YAP5SA
FUW-tetO-HA-YAP5SA/S94A
FUW-tetO-MCS (empty vector)
FUW-tetO-RFP
FUdeltaGW-rtTA
FUW-tetO-N1ICD-Myc
FUW-tetO-GFP-DNMAML1

dependent variables

Cellular outcome(s) influenced by the manipulated factors

control variables

Fibronectin
Cytochalasin D

positive controls

8xGTIIC-lux
3D.A-Lux

negative controls

FUW-tetO-MCS (empty vector)

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!