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polyclonal rabbit anti trim33 (Cell Signaling Technology Inc)

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Cell Signaling Technology Inc polyclonal rabbit anti trim33
Polyclonal Rabbit Anti Trim33, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/polyclonal+rabbit+anti+trim33/pmc04170004-65-11-15?v=Cell+Signaling+Technology+Inc
Average 93 stars, based on 9 article reviews

polyclonal rabbit anti trim33 - by Bioz Stars, 2026-07

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Fig. 1. SUMO modification of TIF1c. (A) HMEC-TR cells were transfected with either TIF1c expression vector alone or in combination with His6-tagged SUMO-1, Ubc9 or PIAS1 expression vectors. Cell lysates were subjected to His6 pull-down assays with Ni-NTA beads and immunoblotted with anti-TIF1c antibody. (B) Cells lysates of HMEC-TR cells were immunoprecipitated (IP) and immunoblotted (IB) as indicated. Protein expression was monitored by immunoblot analysis of total cell extracts (Input). (C) Cell lysates of HMEC-TR cells transfected with TIF1c and SUMO-1 were immunoprecipitated (IP) and immunoblotted (IB) as indicated. (D) HMEC-TR cells were treated or not with TGFb for the indicated times. Total protein extracts were lysed in the presence of 10 mM N-ethylmaleimide to inhibit sumoylase activities and immunoblotted with anti-TIF1c antibody. GAPDH was used as a loading control. (E) HMEC-TR cells were transfected with TIF1c and SUMO-1 and treated with TGFb as indicated. Total protein extracts were immunoblotted with anti-TIF1c antibody. GAPDH loading controls are as above. (F) HMEC-TR cells were treated or not with TGFb for 24 h and then with the proteasome inhibitor MG132 for the indicated time. Total protein extracts were immunoblotted with anti-TIF1c antibody. GAPDH was used as a loading control. (G,H) Cell lysates of HMEC-TR cells transfected with the indicated expression vectors were immunoblotted with anti-TIF1c antibody. SENP1m is a catalytically inactive mutant of SENP1 (R630L/K631M). GAPDH was used as a loading control.

Journal: Journal of cell science

Article Title: TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

doi: 10.1242/jcs.126748

Figure Lengend Snippet: Fig. 1. SUMO modification of TIF1c. (A) HMEC-TR cells were transfected with either TIF1c expression vector alone or in combination with His6-tagged SUMO-1, Ubc9 or PIAS1 expression vectors. Cell lysates were subjected to His6 pull-down assays with Ni-NTA beads and immunoblotted with anti-TIF1c antibody. (B) Cells lysates of HMEC-TR cells were immunoprecipitated (IP) and immunoblotted (IB) as indicated. Protein expression was monitored by immunoblot analysis of total cell extracts (Input). (C) Cell lysates of HMEC-TR cells transfected with TIF1c and SUMO-1 were immunoprecipitated (IP) and immunoblotted (IB) as indicated. (D) HMEC-TR cells were treated or not with TGFb for the indicated times. Total protein extracts were lysed in the presence of 10 mM N-ethylmaleimide to inhibit sumoylase activities and immunoblotted with anti-TIF1c antibody. GAPDH was used as a loading control. (E) HMEC-TR cells were transfected with TIF1c and SUMO-1 and treated with TGFb as indicated. Total protein extracts were immunoblotted with anti-TIF1c antibody. GAPDH loading controls are as above. (F) HMEC-TR cells were treated or not with TGFb for 24 h and then with the proteasome inhibitor MG132 for the indicated time. Total protein extracts were immunoblotted with anti-TIF1c antibody. GAPDH was used as a loading control. (G,H) Cell lysates of HMEC-TR cells transfected with the indicated expression vectors were immunoblotted with anti-TIF1c antibody. SENP1m is a catalytically inactive mutant of SENP1 (R630L/K631M). GAPDH was used as a loading control.

Article Snippet: Mouse monoclonal anti-flag antibody (EL1B11, Euromedex), mouse monoclonal anti-HA antibody (12CA5, Roche), rabbit polyclonal anti-Ubc9 antibody (#10759, SantaCruz), mouse monoclonal anti-Smad4 B8 antibody (#7966, SantaCruz), rabbit polyclonal anti-TIF1c antibody (A301-060A, Bethyl), rabbit polyclonal anti-SUMO-1 antibody (SantaCruz), mouse monoclonal anti-TIF1c antibody (TIF3E9, Euromedex) were used for immunoblotting.

Techniques: Modification, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Mutagenesis

Fig. 2. Identification of TIF1c sumoylation sites. (A) HMEC-TR cells were transfected with His6-tagged SUMO-1 and TIF1c mutants as indicated. Lysates were subjected to a His6 pull-down using Ni-NTA beads and immunoblotted with anti-TIF1c antibody. (B) Schematic representation of TIF1c. The structure is characterized by an N-terminal motif composed of a RING finger and, surrounding the middle domain, two B-box domains (B1 and B2) and a coiled-coil (CC) domain (RBCC/TRIM), and a C-terminal PHD finger (P) and bromodomain (B). The four functional SUMO sites are shown in red. (C) Conservation of the YKx (D/E) sumoylation motifs in TIF1c from various species. The TIF1c sequence harboring the SUMO attachment sites was compared between the indicated species. Four conserved SUMO-targeted lysine residues (K) are shown in red. ‘*’ and ‘:’ denote identical or related amino acids, respectively. (D) Cell lysates from HMEC-TR cells transfected with the indicated constructs were processed as in A. Note that the N-terminal fusion of full-length Ubc9 to TIF1c causes an 18-kDa shift compared with that of endogenous TIF1c. (E) HMEC-TR cells were transfected with FLAG-tagged wild-type (TIF1c-WT) or the sumoylation-defective mutant (TIF1c-Mut) and treated with 20 mg/ml cycloheximide (CHX) for the indicated times. Protein expression was monitored by immunoblot analysis of total cell extracts using anti-FLAG antibody. GAPDH was used as a loading control. (F) Band intensities in E were quantified using a densitometer. Data are representative of two independent experiments. (G) MCF10A cells were stably infected with expression vectors encoding TIF1c-WT or TIF1c-Mut. TIF1c nuclear localization was visualized by immunofluorescence using an anti-TIF1c antibody. Scale bar: 26 mm.

Journal: Journal of cell science

Article Title: TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

doi: 10.1242/jcs.126748

Figure Lengend Snippet: Fig. 2. Identification of TIF1c sumoylation sites. (A) HMEC-TR cells were transfected with His6-tagged SUMO-1 and TIF1c mutants as indicated. Lysates were subjected to a His6 pull-down using Ni-NTA beads and immunoblotted with anti-TIF1c antibody. (B) Schematic representation of TIF1c. The structure is characterized by an N-terminal motif composed of a RING finger and, surrounding the middle domain, two B-box domains (B1 and B2) and a coiled-coil (CC) domain (RBCC/TRIM), and a C-terminal PHD finger (P) and bromodomain (B). The four functional SUMO sites are shown in red. (C) Conservation of the YKx (D/E) sumoylation motifs in TIF1c from various species. The TIF1c sequence harboring the SUMO attachment sites was compared between the indicated species. Four conserved SUMO-targeted lysine residues (K) are shown in red. ‘*’ and ‘:’ denote identical or related amino acids, respectively. (D) Cell lysates from HMEC-TR cells transfected with the indicated constructs were processed as in A. Note that the N-terminal fusion of full-length Ubc9 to TIF1c causes an 18-kDa shift compared with that of endogenous TIF1c. (E) HMEC-TR cells were transfected with FLAG-tagged wild-type (TIF1c-WT) or the sumoylation-defective mutant (TIF1c-Mut) and treated with 20 mg/ml cycloheximide (CHX) for the indicated times. Protein expression was monitored by immunoblot analysis of total cell extracts using anti-FLAG antibody. GAPDH was used as a loading control. (F) Band intensities in E were quantified using a densitometer. Data are representative of two independent experiments. (G) MCF10A cells were stably infected with expression vectors encoding TIF1c-WT or TIF1c-Mut. TIF1c nuclear localization was visualized by immunofluorescence using an anti-TIF1c antibody. Scale bar: 26 mm.

Techniques: Transfection, Functional Assay, Sequencing, Construct, Mutagenesis, Expressing, Western Blot, Control, Stable Transfection, Infection, Immunofluorescence

Fig. 3. TIF1c is SUMO-modified in the nucleus. (A) The TIF1c NLS: basic amino acids are underlined, the mutated lysine (K1118) is shown in bold. (B) TIF1c immunofluorescence in HMEC-TR cells transfected with the indicated constructs. (C) Lysates from HMEC-TR cells transfected with indicated constructs were subjected to His6 pull-down using Ni-NTA beads and immunoblotted with anti-TIF1c antibody. (D) HMEC-TR cells were co- transfected with the pGL3(CAGA)9-Luc reporter vector together with the pRL-SV40 internal control vector as well as with the indicated TIF1c expression vectors. Cells were treated with the TbRI kinase inhibitor SB- 431542 (SB) as control, or with TGFb for 24 hours. SB-431542 blocks any signaling arising from low endogenous TGFb production. Relative luciferase activity is given in arbitrary units as the mean 6 s.d. of an experiment performed in triplicate, representative of three independent experiments. Protein expression (TIF1c, GAPDH) was monitored by immunoblot analysis of total cell extracts.

Journal: Journal of cell science

Article Title: TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

doi: 10.1242/jcs.126748

Figure Lengend Snippet: Fig. 3. TIF1c is SUMO-modified in the nucleus. (A) The TIF1c NLS: basic amino acids are underlined, the mutated lysine (K1118) is shown in bold. (B) TIF1c immunofluorescence in HMEC-TR cells transfected with the indicated constructs. (C) Lysates from HMEC-TR cells transfected with indicated constructs were subjected to His6 pull-down using Ni-NTA beads and immunoblotted with anti-TIF1c antibody. (D) HMEC-TR cells were co- transfected with the pGL3(CAGA)9-Luc reporter vector together with the pRL-SV40 internal control vector as well as with the indicated TIF1c expression vectors. Cells were treated with the TbRI kinase inhibitor SB- 431542 (SB) as control, or with TGFb for 24 hours. SB-431542 blocks any signaling arising from low endogenous TGFb production. Relative luciferase activity is given in arbitrary units as the mean 6 s.d. of an experiment performed in triplicate, representative of three independent experiments. Protein expression (TIF1c, GAPDH) was monitored by immunoblot analysis of total cell extracts.

Techniques: Modification, Immunofluorescence, Transfection, Construct, Plasmid Preparation, Control, Expressing, Luciferase, Activity Assay, Western Blot

Fig. 4. Lack of sumoylation compromises the ability of TIF1c to inhibit Smad complex formation. (A–G) HMEC-TR cells were transfected with the indicated vectors. At 48 h post-transfection, cells were treated or not with TGFb for 2 h. Lysates were immunoprecipitated (IP) and immunoblotted (IB) as indicated. Protein expression was monitored by immunoblot analysis of total cell extracts (Input). (H) Sumoylation does not affect TIF1c E3 ubiquitin ligase activity on Smad4. HMEC-TR cells were transfected with the indicated vectors. At 48 h post-transfection, cell lysates were immunoprecipitated (IP) with anti- FLAG antibody and immunoblotted (IB) with anti-HA antibody to detect Smad4 ubiquitylation. Protein expression was monitored by immunoblot analysis of total cell extracts (Input). Ub, ubiquitin.

Journal: Journal of cell science

Article Title: TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

doi: 10.1242/jcs.126748

Figure Lengend Snippet: Fig. 4. Lack of sumoylation compromises the ability of TIF1c to inhibit Smad complex formation. (A–G) HMEC-TR cells were transfected with the indicated vectors. At 48 h post-transfection, cells were treated or not with TGFb for 2 h. Lysates were immunoprecipitated (IP) and immunoblotted (IB) as indicated. Protein expression was monitored by immunoblot analysis of total cell extracts (Input). (H) Sumoylation does not affect TIF1c E3 ubiquitin ligase activity on Smad4. HMEC-TR cells were transfected with the indicated vectors. At 48 h post-transfection, cell lysates were immunoprecipitated (IP) with anti- FLAG antibody and immunoblotted (IB) with anti-HA antibody to detect Smad4 ubiquitylation. Protein expression was monitored by immunoblot analysis of total cell extracts (Input). Ub, ubiquitin.

Techniques: Transfection, Immunoprecipitation, Expressing, Western Blot, Ubiquitin Proteomics, Activity Assay

Fig. 5. TIF1c sumoylation restricts the residence of Smad4 on the PAI-1 promoter. HMEC-TR cells were transfected with either empty vector (A), FLAG- TIF1c-WT (B) or FLAG-TIF1c-Mut (C) and treated with SB-431542 (SB) or TGFb for the indicated time. ChIP assays were carried out using anti-Smad4, anti- TIF1c or anti-FLAG antibodies as indicated. Precipitated genomic DNA was subjected to quantitative PCR to amplify the PAI-1 promoter region harboring the Smad-binding elements. (D) HMEC-TR cells were transfected with either empty vector, TIF1c-WT or Ubc9-TIF1c and treated with SB-431542 or TGFb for the indicated time (minutes). ChIP assays were carried out using anti-Smad4 antibody. Precipitated genomic DNA was subjected to quantitative PCR to amplify the PAI-1 promoter region harboring the Smad-binding elements. In all panels, results are shown as the percentage of input values. Error bars represent s.d. from triplicate samples, and each panel is representative of three independent experiments.

Journal: Journal of cell science

Article Title: TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

doi: 10.1242/jcs.126748

Figure Lengend Snippet: Fig. 5. TIF1c sumoylation restricts the residence of Smad4 on the PAI-1 promoter. HMEC-TR cells were transfected with either empty vector (A), FLAG- TIF1c-WT (B) or FLAG-TIF1c-Mut (C) and treated with SB-431542 (SB) or TGFb for the indicated time. ChIP assays were carried out using anti-Smad4, anti- TIF1c or anti-FLAG antibodies as indicated. Precipitated genomic DNA was subjected to quantitative PCR to amplify the PAI-1 promoter region harboring the Smad-binding elements. (D) HMEC-TR cells were transfected with either empty vector, TIF1c-WT or Ubc9-TIF1c and treated with SB-431542 or TGFb for the indicated time (minutes). ChIP assays were carried out using anti-Smad4 antibody. Precipitated genomic DNA was subjected to quantitative PCR to amplify the PAI-1 promoter region harboring the Smad-binding elements. In all panels, results are shown as the percentage of input values. Error bars represent s.d. from triplicate samples, and each panel is representative of three independent experiments.

Techniques: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay

Fig. 6. TIF1c sumoylation regulates TGFb- induced EMT processes. (A) HMEC-TR cells cotransfected with the pGL3(CAGA)9-Luc reporter vector together with increasing amounts of either wild-type (TIF1c-WT) or mutated (TIF1c-Mut) TIF1c were treated with TGFb for 24 h before measurement of luciferase activities. Error bars represent s.d. (n53). Expression levels of TIF1c and GAPDH proteins are shown. (B–D) Stably infected MCF10A cells expressing wild-type (TIF1c-WT) or mutated (TIF1c-Mut) TIF1c were treated with SB-431542 (SB) or TGFb for 24 h (B) or 96 h (C,D). (B) Expression of the mRNA encoding PAI-1 (serpine 1), CDH2 (N- cadherin) and CDH11 (OB-cadherin) was determined by RT-qPCR. Values were normalized to the amount of mRNA for HPRT and expressed relative to the value obtained in TGFb-untreated controls (expressed as fold induction by TGFb). Error bars represent s.d. (n53). *P,0.05; **P,0.01. (C) E-cadherin expression was monitored by immunofluorescence. DAPI was used for nuclear staining. (D) The subcellular localization of actin was detected by imaging phalloidin–TRITC. Scale bars: 26 mm. (E) Stably transfected MCF10A cells inactivated for TIF1c (sh-TIF1c) or expressing WT (TIF1c-WT) or mutated TIF1c (TIF1c-Mut) were treated with SB- 431542 (SB) or TGFb for 48 h prior to perform the Boyden chamber migration assay. 5% serum was used as a chemo-attractant during 22 h. Migrating cells were stained with Calcein AM and counted from random fields. Error bars represent s.d. The experiment shown is representative of three separate experiments performed in triplicate. *P,0.05; **P,0.01; ***P,0.001.

Journal: Journal of cell science

Article Title: TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling.

doi: 10.1242/jcs.126748

Figure Lengend Snippet: Fig. 6. TIF1c sumoylation regulates TGFb- induced EMT processes. (A) HMEC-TR cells cotransfected with the pGL3(CAGA)9-Luc reporter vector together with increasing amounts of either wild-type (TIF1c-WT) or mutated (TIF1c-Mut) TIF1c were treated with TGFb for 24 h before measurement of luciferase activities. Error bars represent s.d. (n53). Expression levels of TIF1c and GAPDH proteins are shown. (B–D) Stably infected MCF10A cells expressing wild-type (TIF1c-WT) or mutated (TIF1c-Mut) TIF1c were treated with SB-431542 (SB) or TGFb for 24 h (B) or 96 h (C,D). (B) Expression of the mRNA encoding PAI-1 (serpine 1), CDH2 (N- cadherin) and CDH11 (OB-cadherin) was determined by RT-qPCR. Values were normalized to the amount of mRNA for HPRT and expressed relative to the value obtained in TGFb-untreated controls (expressed as fold induction by TGFb). Error bars represent s.d. (n53). *P,0.05; **P,0.01. (C) E-cadherin expression was monitored by immunofluorescence. DAPI was used for nuclear staining. (D) The subcellular localization of actin was detected by imaging phalloidin–TRITC. Scale bars: 26 mm. (E) Stably transfected MCF10A cells inactivated for TIF1c (sh-TIF1c) or expressing WT (TIF1c-WT) or mutated TIF1c (TIF1c-Mut) were treated with SB- 431542 (SB) or TGFb for 48 h prior to perform the Boyden chamber migration assay. 5% serum was used as a chemo-attractant during 22 h. Migrating cells were stained with Calcein AM and counted from random fields. Error bars represent s.d. The experiment shown is representative of three separate experiments performed in triplicate. *P,0.05; **P,0.01; ***P,0.001.

Techniques: Plasmid Preparation, Luciferase, Expressing, Stable Transfection, Infection, Quantitative RT-PCR, Immunofluorescence, Staining, Imaging, Transfection, Migration

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