gst yap  (Novus Biologicals)

Journal: BBA Advances

Article Title: Narciclasine is a novel YAP inhibitor that disturbs interaction between YAP and TEAD4

doi: 10.1016/j.bbadva.2021.100008

Figure Lengend Snippet: Interaction of narciclasine with YAP. (A) Schematic structure of human YAP and TAZ. WW, WW domain; CC, coiled-coil domain. 127Serine (127S) in YAP and 89Serine (89S) in TAZ are the main phosphorylation sites by Lats1/2. (B) Structure of narciclasine. (C) Interaction of narciclasine with YAP. Interaction between narciclasine and GST-YAP was measured by SPR. GST-YAP and GST alone were captured at 8,000 and 2,900 RU, respectively. Solid line, GST-YAP; broken line, GST alone. The Kd of narciclasine to GST-YAP was 970 μM. Significant differences between GST alone and GST-YAP are indicated with asterisks.

Article Snippet: GST fusion proteins Human YAP was subcloned into a pGEX-4T-1 vector (GE Healthcare).

Techniques:

Journal: BBA Advances

Article Title: Narciclasine is a novel YAP inhibitor that disturbs interaction between YAP and TEAD4

doi: 10.1016/j.bbadva.2021.100008

Figure Lengend Snippet: Determination of the narciclasine-interacting region in YAP. (A) Schematic structures of GST fusion protein used. (B, C) Binding ability of narciclasine to the N-terminal part of YAP (B), but not to its C-terminal part (C). Binding capacity of narciclasine to each GST fusion protein was measured using SPR. As a negative control, GST alone was used. (B) GST-YAP-N and GST alone were captured at 6,100 and 2,300 RU, respectively. The Kd of narciclasine to GST-YAP-N was 245 μM. (C) GST-YAP-C and GST alone were captured at 12,000 and 5,600 RU, respectively. Significant differences between GST alone and GST-YAP-N or GST-YAP-C are indicated with asterisks.

Article Snippet: GST fusion proteins Human YAP was subcloned into a pGEX-4T-1 vector (GE Healthcare).

Techniques: Binding Assay, Negative Control

Journal: BBA Advances

Article Title: Narciclasine is a novel YAP inhibitor that disturbs interaction between YAP and TEAD4

doi: 10.1016/j.bbadva.2021.100008

Figure Lengend Snippet: Inhibition of YAP-mediated transcriptional activity by narciclasine. (A) Inhibitory effect of narciclasine on the activity of CTGF-luc. 293A cells were transfected with CTGF-luc in the presence or absence of YAP. Significant differences from the control with YAP are indicated with asterisks. VP, verteporfin. (B) The expressions of YAP and β-actin in total lysates from cells transfected with CTGF-luc. Anti-Flag (upper panel) and anti-β-actin antibodies (lower panel) were used to detect proteins. VP, verteporfin. (C) Inhibitory effect of narciclasine on the activity of 5xUAS-luc. 293A cells were transfected with 5xUAS and Gal4-TEAD4 in the presence or absence of YAP. Significant differences from the control with YAP are indicated with asterisks. VP, verteporfin. (D) The expressions of YAP and β-actin in total lysates from cells transfected with 5xUAS and Gal4-TEAD4. Anti-Flag (upper panel) and anti-β-actin antibodies (lower panel) were used to detect proteins. VP, verteporfin. (E) Inhibition of endogenous CTGF mRNA expression by narciclasine. NCI-H290 cells were treated with VP or narciclasine for 24 h. CTGF mRNAs were measured by qPCR. Significant differences from the control are indicated with asterisks. VP, verteporfin. (F) Competition of narciclasine with TEAD4 for binding to YAP. Different amounts of Myc-TEAD4 were incubated with GST-YAP in the absence or presence of narciclasine. Then, a GST-pulldown assay was performed. Upper panel, Myc-TEAD4 pulled down with GST-YAP; middle panel, expression of total Myc-TEAD4; lower panel, expression of GST and GST-YAP with Ponceau S staining.

Article Snippet: GST fusion proteins Human YAP was subcloned into a pGEX-4T-1 vector (GE Healthcare).

Techniques: Inhibition, Activity Assay, Transfection, Expressing, Binding Assay, Incubation, GST Pulldown Assay, Staining

Journal: Nature Communications

Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

doi: 10.1038/s41467-018-04620-y

Figure Lengend Snippet: SKP2 interacts with YAP and induces its K63-linked polyubiquitination. a The HEK293T SFB-YAP stable cell line was transfected with MYC-tagged E3 ligases, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against FLAG and MYC. b The HEK293T SFB-YAP stable cell line was co-transfected with the K63-specific mutant of HA-ubiquitin and the indicated E3 ligase, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. c Co-immunoprecipitation of endogenous SKP2 with endogenous YAP. d In vitro binding of bacterially purified MBP-SKP2 to in vitro translated and purified GST-YAP. e In vitro binding of bacterially purified MBP-SKP2 to SFB-YAP purified from the HEK293T SFB-YAP stable cell line. MBP-SKP2 or MBP protein was incubated with SFB-YAP protein with or without CIP treatment at 37 °C for 1 h, followed by immunoblotting with antibodies against SKP2, p-YAP (S127), and FLAG. f The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48R or K63R) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. g The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48-specific or K63-specific) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. h Total K63-linkage specific ubiquitinated proteins in control or SKP2 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin. i siRNA targeting CUL1 or SKP1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours after siRNA transfection, cells were transfected with HA-ubiquitin and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j The HEK293T UBCH5c shRNA stable cell line was transfected with SFB-YAP and HA-ubiquitin with or without MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. k Purified GST-YAP protein was incubated with ATP, E1, UBCH5c, and the K63-specific mutant of His-ubiquitin with or without the SCF SKP2 complex, followed by immunoprecipitation with a YAP-specific antibody and immunoblotting with antibodies against ubiquitin and YAP

Article Snippet: For pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (Millipore, 69704) at 4 °C for 2 h. For in vitro binding assays, purified GST-YAP (Novus Biologicals, H00010413-P01) was incubated with purified MBP-SKP2, and purified His-YAP (MyBioSource, MBS717875) was incubated with purified GST-OTUD1, followed by pulldown with Glutathione Sepharose beads (GE healthcare, 17-0756-01).

Techniques: Stable Transfection, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, In Vitro, Binding Assay, Purification, Incubation, Knock-Out, Generated, CRISPR, shRNA

Journal: Nature Communications

Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

doi: 10.1038/s41467-018-04620-y

Figure Lengend Snippet: OTUD1 interacts with YAP and antagonizes its K63-linked ubiquitination. a SFB-tagged DUBs were co-transfected with MYC-YAP into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. b Seven SFB-DUBs were co-transfected with MYC-YAP and HA-ubiquitin into HEK293T cells, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. c The HEK293T SFB-YAP stable cell line was transfected with four YAP-interacting DUBs along with an 8× GTIIC luciferase reporter and a TK-Renilla luciferase reporter. Reporter activity was measured 48 h after transfection. Error bars are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. n = 3 biological replicates. d Co-immunoprecipitation of endogenous OTUD1 with endogenous YAP. e In vitro binding of purified GST-OTUD1 to purified His-YAP. f HEK293T cells were co-transfected with SFB-OTUD1 (wild-type, C320S or H431R), HA-ubiquitin and MYC-YAP, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. g Ubiquitinated SFB-YAP was purified with S-protein beads and incubated with His-OTUD1 (wild-type or C320S) purified from bacteria. After the in vitro deubiquitination reaction, the bound proteins were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against HA, FLAG, and OTUD1. h HEK293T cells were co-transfected with SFB-OTUD1, HA-ubiquitin (wild-type, K48R or K63R) and MYC-YAP, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. i siRNA targeting OTUD1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours later, cells were transfected with HA-ubiquitin (wild-type, K48R or K63R), followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j Total K63-linkage specific ubiquitinated proteins in control or OTUD1 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin

Article Snippet: For pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (Millipore, 69704) at 4 °C for 2 h. For in vitro binding assays, purified GST-YAP (Novus Biologicals, H00010413-P01) was incubated with purified MBP-SKP2, and purified His-YAP (MyBioSource, MBS717875) was incubated with purified GST-OTUD1, followed by pulldown with Glutathione Sepharose beads (GE healthcare, 17-0756-01).

Techniques: Transfection, Western Blot, Immunoprecipitation, Stable Transfection, Luciferase, Activity Assay, Two Tailed Test, In Vitro, Binding Assay, Purification, Incubation, Bacteria, Knock-Out, Generated, CRISPR