Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains

doi: 10.1371/journal.ppat.1005584

Figure Lengend Snippet: (A) IKK complex purified from 293F/NEMO-FLAG knock-in cells was incubated with recombinant E1, Ub, UbcH7 WT or C86A mutant and Tax in ATP buffer at 37°C for 2 h. IKK activation was determined by phosphorylation of IKKα and IKKβ. Polyubiquitin chains were detected by anti-ubiquitin immunoblotting. IKKα, IKKβ and NEMO were also blotted. (B) The same assay as in (A) was performed except the UbcH7 was replaced by UbcH5c or its enzymatic-dead mutant C85A.

Article Snippet: Rabbit antibodies against IKKα (sc-7218, dilution 1:1,000), IKKβ (sc-7607, dilution 1:1,000), NEMO (sc-8330, dilution 1:1,000) and mouse antibody against ubiquitin (sc-8017, dilution 1:1,000) were obtained from Santa Cruz Biotechnology; antibodies against TAK1 (ab109526, dilution 1:1,000), UbcH7 (ab108936, dilution 1:2,000), Ubc13 (ab109286, dilution 1:5,000), RNF8 (ab131221, dilution 1:1,000), p-IKKα S176 (ab138426, dilution 1:500) were from Abcam; antibodies against CYLD (#8462, dilution 1:1,000), p-TAK1 Thr187 (#4536, dilution 1:1,000), p-IKKα/β Ser176/180 (#2697, dilution 1:1,000) and p-IκBα Ser32/36 (#9246, dilution 1:1,000) were from Cell Signaling; antibodies against FLAG-tag (M20008, dilution 1:5,000) and His-tag (M20001, dilution 1:1,000) were from Abmart; antibodies against IκBα and UbcH5c were homemade; and antibody against Tax (mAB, clone Lt-4) was described in Lee et al. [ ].

Techniques: Purification, Knock-In, Incubation, Recombinant, Mutagenesis, Activation Assay, Western Blot

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains

doi: 10.1371/journal.ppat.1005584

Figure Lengend Snippet: (A) In vitro ubiquitination assay with Tax. Different concentrations of Tax protein were incubated with E1, Ub and indicated E2s in ATP buffer at 37°C for 2 h. The products were immunoblotted by anti-ubiquitin and anti-Tax antibodies. (B) The reaction mixtures in (A) by UbcH5c and Tax were incubated with Ni-NTA beads at 4°C for 2 h to immunodeplete His-tagged reaction components (E1, UbcH5c and Tax). The supernatant was immunoblotted by anti-ubiquitin and anti-Tax antibodies. (C) Ubiquitin discharging from Ub~E2s by Tax. Ub~E2 thioester intermediates (prepared as described in ) were incubated with Tax at 30°C for 15 min. Discharged E2s were detected by immunoblotting with their respective antibodies. FH: FLAG-10xHis tag.

Article Snippet: Rabbit antibodies against IKKα (sc-7218, dilution 1:1,000), IKKβ (sc-7607, dilution 1:1,000), NEMO (sc-8330, dilution 1:1,000) and mouse antibody against ubiquitin (sc-8017, dilution 1:1,000) were obtained from Santa Cruz Biotechnology; antibodies against TAK1 (ab109526, dilution 1:1,000), UbcH7 (ab108936, dilution 1:2,000), Ubc13 (ab109286, dilution 1:5,000), RNF8 (ab131221, dilution 1:1,000), p-IKKα S176 (ab138426, dilution 1:500) were from Abcam; antibodies against CYLD (#8462, dilution 1:1,000), p-TAK1 Thr187 (#4536, dilution 1:1,000), p-IKKα/β Ser176/180 (#2697, dilution 1:1,000) and p-IκBα Ser32/36 (#9246, dilution 1:1,000) were from Cell Signaling; antibodies against FLAG-tag (M20008, dilution 1:5,000) and His-tag (M20001, dilution 1:1,000) were from Abmart; antibodies against IκBα and UbcH5c were homemade; and antibody against Tax (mAB, clone Lt-4) was described in Lee et al. [ ].

Techniques: In Vitro, Ubiquitin Assay, Incubation, Western Blot

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains

doi: 10.1371/journal.ppat.1005584

Figure Lengend Snippet: (A) Ubiquitin mutants impair IKK activation by Tax. Jurkat T S100 was incubated with Tax and the indicated ubiquitin mutants in ATP buffer at 30°C for 1 h. IKK activation was detected by phosphorylation of IκBα. (B) Jurkat T S100 was incubated with Tax and different concentrations of CYLD or its enzymatic-dead mutant C601A. IKK activation was detected by phosphorylation of IκBα. (C) The same assay as in (B) was performed except CYLD was replaced by vOTU or its enzymatic-dead mutant C40A. (D) Interaction between NEMO and ubiquitin chains is required for Tax-dependent IKK activation. Cell extracts (S100) of NEMO-deficient Jurkat T (C2) cells were incubated with Tax and NEMO WT or the indicated mutants (100 nM each) in ATP buffer at 30°C for 1 h. IKK activation was detected by phosphorylation of IKKβ and its substrate IκBα. (E) Mixed-linkage polyUb chains synthesized by Tax activate IKK directly. Purified IKK complex was incubated with the indicated polyUb chains (prepared and purified as described in ) in ATP buffer at 37°C for 2 h. IKK activation was detected by phosphorylation of IKKβ. Mixed: Mixed linkage polyUb chains synthesized by Tax and UbcH5c. K63: K63 linkage polyUb chains synthesized by TRAF6 and Ubc13/Uev2. K48: K48 linkage polyUb chains synthesized by IpaH and UbcH5c. Linear: Linear linkage polyUb chains synthesized by HOIP-RBRC and UbcH7.

Article Snippet: Rabbit antibodies against IKKα (sc-7218, dilution 1:1,000), IKKβ (sc-7607, dilution 1:1,000), NEMO (sc-8330, dilution 1:1,000) and mouse antibody against ubiquitin (sc-8017, dilution 1:1,000) were obtained from Santa Cruz Biotechnology; antibodies against TAK1 (ab109526, dilution 1:1,000), UbcH7 (ab108936, dilution 1:2,000), Ubc13 (ab109286, dilution 1:5,000), RNF8 (ab131221, dilution 1:1,000), p-IKKα S176 (ab138426, dilution 1:500) were from Abcam; antibodies against CYLD (#8462, dilution 1:1,000), p-TAK1 Thr187 (#4536, dilution 1:1,000), p-IKKα/β Ser176/180 (#2697, dilution 1:1,000) and p-IκBα Ser32/36 (#9246, dilution 1:1,000) were from Cell Signaling; antibodies against FLAG-tag (M20008, dilution 1:5,000) and His-tag (M20001, dilution 1:1,000) were from Abmart; antibodies against IκBα and UbcH5c were homemade; and antibody against Tax (mAB, clone Lt-4) was described in Lee et al. [ ].

Techniques: Activation Assay, Incubation, Mutagenesis, Synthesized, Purification

Journal: PLoS Pathogens

Article Title: HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains

doi: 10.1371/journal.ppat.1005584

Figure Lengend Snippet: (A) Ubiquitin R63 mutant impairs IKK activation by Tax. The same assay as in was performed except ubiquitin was replaced by Ub R48 or R63 mutants. (B) Polyubiquitin chains pre-synthesized by E1, UbcH5c, Tax and Ub WT or the indicated mutants were incubated with purified IKK complex, in ATP buffer at 37°C for 2 h. IKK activation was detected by phosphorylation of IKKβ. (C) Ub R63 is inefficient in polyubiquitination by Tax. Tax was incubated with E1, UbcH5c, and the indicated Ub in ATP buffer at 37°C for the indicated time. Synthesis of polyUb chains was determined by immunoblotting using anti-Ub antibody. (D) Model illustrating IKK activation by Tax.

Article Snippet: Rabbit antibodies against IKKα (sc-7218, dilution 1:1,000), IKKβ (sc-7607, dilution 1:1,000), NEMO (sc-8330, dilution 1:1,000) and mouse antibody against ubiquitin (sc-8017, dilution 1:1,000) were obtained from Santa Cruz Biotechnology; antibodies against TAK1 (ab109526, dilution 1:1,000), UbcH7 (ab108936, dilution 1:2,000), Ubc13 (ab109286, dilution 1:5,000), RNF8 (ab131221, dilution 1:1,000), p-IKKα S176 (ab138426, dilution 1:500) were from Abcam; antibodies against CYLD (#8462, dilution 1:1,000), p-TAK1 Thr187 (#4536, dilution 1:1,000), p-IKKα/β Ser176/180 (#2697, dilution 1:1,000) and p-IκBα Ser32/36 (#9246, dilution 1:1,000) were from Cell Signaling; antibodies against FLAG-tag (M20008, dilution 1:5,000) and His-tag (M20001, dilution 1:1,000) were from Abmart; antibodies against IκBα and UbcH5c were homemade; and antibody against Tax (mAB, clone Lt-4) was described in Lee et al. [ ].

Techniques: Mutagenesis, Activation Assay, Synthesized, Incubation, Purification, Western Blot

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: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Stable Transfection, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Ubiquitin Proteomics, In Vitro, Binding Assay, Purification, Incubation, Control, 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: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

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

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: K63-linked ubiquitination promotes YAP nuclear localization and activity. a HEK293T cells were transfected with SFB-YAP and HA-ubiquitin (wild-type, K48 or K48R) and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. H: high density; L: low density. b HEK293A cells were stably transfected with SFB-YAP and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against K63-linkage-specific polyubiquitin, p-YAP (S127) and FLAG. H: high density; L: low density. c Total K63-linkage specific ubiquitinated proteins in MCF10A cells were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin. H: high density; L: low density. d HEK293T cells were transfected with SFB-YAP (wild-type, K321R, K497R, or K321R/K497R) 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. e Upper panel: YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (wild-type or K321R/K497R) and immunostained with a YAP-specific antibody (green). DAPI (blue) was used to stain DNA. Lower panel: immunoblotting of YAP (left) and quantification of immunofluorescence data (right). Statistical significance was determined by a chi-square test. * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar, 20 μm. f Luciferase activity in HEK293T cells co-transfected with SFB-YAP (wild-type or K321R/K497R), an 8×GTIIC luciferase reporter and a TK-Renilla luciferase reporter. n = 3 biological replicates. g qPCR of ANKRD1 , CTGF , and CYR61 in cells described in f . n = 3 biological replicates. Error bars in f and 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

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Activity Assay, Transfection, Western Blot, Stable Transfection, Immunoprecipitation, Mutagenesis, Knock-Out, Generated, CRISPR, Staining, Immunofluorescence, Luciferase, Two Tailed Test

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: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Stable Transfection, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Ubiquitin Proteomics, In Vitro, Binding Assay, Purification, Incubation, Control, 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: SKP2 promotes nuclear localization and transcriptional activity of YAP. a , b HEK293A cells were stably transfected with MYC-SKP2, cultured at medium ( a ) or high ( b ) density, and immunostained with a YAP-specific antibody (green). DAPI (blue) was used to stain DNA. Scale bar, 20 μm. c , d HEK293A cells expressing SKP2 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 HEK293A cells were stably infected with HA-FLAG-YAP and SKP2 shRNA and immunostained with an HA-specific antibody (red). DAPI (blue) was used to stain DNA. Scale bar, 20 μm. f YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (WT or 2KR) with or without MYC-SKP2, and immunostained with antibodies against YAP (red) and MYC (green). DAPI (blue) was used to stain DNA. Scale bar, 20 μm. g – i qPCR of ANKRD1 , CYR61 , and CTGF in HEK293A cells expressing MYC-SKP2 ( g ), SKP2 shRNA ( h ) or SKP2 gRNA ( i , upper panel). Lower panel in i : immunoblotting of SKP2 and α-tubulin in three independent SKP2 knockout HEK293A clones generated by CRISPR-Cas9. n = 3 biological replicates. Error bars in g – i 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

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Activity Assay, Stable Transfection, Transfection, Cell Culture, Staining, Expressing, shRNA, Infection, Knock-Out, Generated, CRISPR, Western Blot, Clone Assay, Two Tailed Test

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: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

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

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 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

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Activity Assay, Western Blot, Stable Transfection, Transfection, Staining, Expressing, shRNA, Knock-Out, Clone Assay, Generated, CRISPR, Two Tailed Test

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: 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

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Transfection, Knock-Out, Generated, CRISPR, Staining, Stable Transfection, Expressing, shRNA, Control, Mutagenesis, Plasmid Preparation

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 and K63-linked ubiquitination enhance the YAP-TEAD interaction. a HEK293A cells were stably transfected with SFB-YAP and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. H: high density; L: low density. b The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2 and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. c The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2. SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. d HEK293T cells were transfected with SFB-YAP (WT or the 2KR mutant). SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. e SKP2 forms a complex with YAP and TEAD1. HA-TEAD1 and MYC-SKP2 were co-transfected with or without SFB-YAP into HEK293T cells. SFB-YAP was pulled down with streptavidin beads and then eluted with biotin buffer. The eluded proteins were subjected to immunoprecipitation with a MYC-specific antibody to pull down MYC-SKP2 and immunoblotting with antibodies against FLAG, HA, and MYC

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Stable Transfection, Transfection, Western Blot, Incubation, Purification, Bacteria, In Vitro, Binding Assay, Mutagenesis, Immunoprecipitation

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: 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

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Western Blot, Transduction, Mutagenesis, Injection, shRNA, Expressing, Activity Assay, Two Tailed Test

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 and K63-linked ubiquitination enhance the YAP-TEAD interaction. a HEK293A cells were stably transfected with SFB-YAP and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. H: high density; L: low density. b The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2 and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. c The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2. SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. d HEK293T cells were transfected with SFB-YAP (WT or the 2KR mutant). SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. e SKP2 forms a complex with YAP and TEAD1. HA-TEAD1 and MYC-SKP2 were co-transfected with or without SFB-YAP into HEK293T cells. SFB-YAP was pulled down with streptavidin beads and then eluted with biotin buffer. The eluded proteins were subjected to immunoprecipitation with a MYC-specific antibody to pull down MYC-SKP2 and immunoblotting with antibodies against FLAG, HA, and MYC

Article Snippet: Antibodies against YAP (14074, 1:1000), phospho-YAP (4911, 1:500), SKP2 (2652, 1:1000), UBCH5c (4330, 1:500), GST (2624, 1:1000), Angiomotin (43130, 1:1000), TEAD (13295, 1:500), 14-3-3 (8312, 1:500), RBX1 (11922, 1:500), and LATS1 (3477, 1:500) were from Cell Signaling Technology.

Techniques: Ubiquitin Proteomics, Stable Transfection, Transfection, Western Blot, Incubation, Purification, Bacteria, In Vitro, Binding Assay, Mutagenesis, Immunoprecipitation