Fig 1: 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
Fig 2: K63-linked ubiquitination of YAP is independent of Hippo signaling-mediated YAP phosphorylation. a Immunoblotting of p-YAP (S127), FLAG-YAP, and GAPDH in HEK293T cells transfected with SFB-YAP (wild-type, K321R, K497R, or K321R/K497R). b YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (S127A or S127A/K321R/K497R) and immunostained with a YAP-specific antibody (green). DAPI (blue) was used to stain DNA. Two different fields are shown. Scale bar, 20 µm. c Immunoblotting of p-YAP (S127), YAP, and SKP2 in HEK293A cells stably expressing MYC-SKP2 (upper panel) or SKP2 shRNA ( lower panel). Scr: a scramble control. d Immunoblotting of p-YAP (S127), YAP, OTUD1, and GAPDH in HEK293A cells stably transfected with HA-OTUD1 (upper panel) or OTUD1 shRNA (lower panel). Scr: a scramble control. e HEK293T cells were co-transfected with SFB-YAP (wild-type or S127A), MYC-SKP2 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. f HEK293T cells were co-transfected SFB-YAP (wild-type or S127A), FLAG-OTUD1 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. g YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (WT or S127A) with or without SKP2 shRNA (on a GFP-positive pGIPZ vector), and immunostained with a YAP-specific antibody (red). DAPI (blue) was used to stain DNA. Scr: a scramble control. Scale bar, 20 µm
Fig 3: OTUD1 inhibits nuclear localization and transcriptional activity of YAP. a Upper panel: immunoblotting of OTUD1 and GAPDH in HEK293A, BT549, and LM2 cell lines. Lower panel: immunoblotting of FLAG-OTUD1 and GAPDH in LM2 cells stably transfected with SFB-OTUD1. b LM2 cells stably overexpressing OTUD1 were immunostained with a YAP-specific antibody (red). DAPI (blue) was used to stain DNA. Scale bar, 20 µm. c, d HEK293A cells expressing OTUD1 shRNA (c) or gRNA (d) were immunostained with a YAP-specific antibody (red in c; green in d). DAPI (blue) was used to stain DNA. Scale bar, 20 µm. e–g qPCR of ANKRD1, CTGF, and CYR61 in BT549 cells expressing HA-OTUD1 (e) and in HEK293A cells expressing OTUD1 shRNA (f) or OTUD1 gRNA (g, left panel). Right panel in g: immunoblotting of OTUD1 and GAPDH in two independent OTUD1 knockout HEK293A clones generated by CRISPR-Cas9. n = 3 biological replicates. Error bars in e–g 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
Fig 4: K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and GAPDH in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in (a). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d, e Tumor growth curves (d) and tumor weight (at the endpoint, e) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, SKP2, and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h, i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 (n = 3951 patients, probe: 203625_x_at, (h) or OTUD1 (n = 1764 patients, probe: 226140_s_at, (i) expression levels. Data were obtained from http://kmplot.com/analysis/. Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a, b, and d–g 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
Supplier Page from MilliporeSigma for Anti-OTUD1 antibody produced in rabbit