Journal: Oncotarget

Article Title: Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is associated with immune phases of patients with chronic hepatitis B

doi: 10.18632/oncotarget.15683

Figure Lengend Snippet: A . The relative expression of TIPE2 mRNA in peripheral blood mononuclear cells in all the patients and healthy controls. B . The mRNA expression levels of IL-6, IL-10, TNF-α, and IFN-γ in all the patients and healthy controls. C . Expression of TIPE2 mRNA from peripheral blood mononuclear cells in healthy controls and CHB patients with IT, IC, LR, and ENH. D . The mRNA expression levels of IL-6, IL-10, TNF-α, and IFN-γ in healthy controls and CHB patients with IT, IC, LR and ENH. E . Expression of TIPE2 mRNA in CHB patients stratified by the statue of HBeAg and HBVDNA. F. ROC analysis of the predicative accuracy for TIPE2 mRNA in discriminating IC from IT and ENH from LR.

Article Snippet: After PBS washing, the slides were incubated using rabbit anti-TIPE2 polyclonal antibody (dilution 1:50, ProteinTech Group (Chicago, IL, USA) at 4°C for overnight and then polymer Helper or poly-HRP anti-rabbit IgG for 20 min at 37°C.

Techniques: Expressing

Journal: Oncotarget

Article Title: Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is associated with immune phases of patients with chronic hepatitis B

doi: 10.18632/oncotarget.15683

Figure Lengend Snippet: A .- D . The positive staining for TIPE2 was found mainly in the hepatocytes (200×). E . Relative mean density analysis showed the difference in hepatic TIPE2 staining between CHB group and healthy controls. F . Expression of hepatic TIPE2 staining between inflammation grade 0, grade (1-2) and grade (3-4). G . Expression of hepatic TIPE2 staining between fibrosis stage (3-4), stage (1-2), and fibrosis stage 0.

Article Snippet: After PBS washing, the slides were incubated using rabbit anti-TIPE2 polyclonal antibody (dilution 1:50, ProteinTech Group (Chicago, IL, USA) at 4°C for overnight and then polymer Helper or poly-HRP anti-rabbit IgG for 20 min at 37°C.

Techniques: Staining, Expressing

Journal: Oncotarget

Article Title: Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is associated with immune phases of patients with chronic hepatitis B

doi: 10.18632/oncotarget.15683

Figure Lengend Snippet: Correlations between TIPE2 mRNA levels and other parameters using univariate and multivariate analysis in all the patients and healthy controls

Article Snippet: After PBS washing, the slides were incubated using rabbit anti-TIPE2 polyclonal antibody (dilution 1:50, ProteinTech Group (Chicago, IL, USA) at 4°C for overnight and then polymer Helper or poly-HRP anti-rabbit IgG for 20 min at 37°C.

Techniques:

Journal: Oncotarget

Article Title: Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is associated with immune phases of patients with chronic hepatitis B

doi: 10.18632/oncotarget.15683

Figure Lengend Snippet: Primers sequence used for real-time RT-PCR

Article Snippet: After PBS washing, the slides were incubated using rabbit anti-TIPE2 polyclonal antibody (dilution 1:50, ProteinTech Group (Chicago, IL, USA) at 4°C for overnight and then polymer Helper or poly-HRP anti-rabbit IgG for 20 min at 37°C.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: TIPE2 serves as a favorable prognostic biomarker and suppresses cholangiocarcinoma progression by targeting RAC1-mediated integrin αvβ6 trafficking

doi: 10.1038/s41598-026-37540-9

Figure Lengend Snippet: The expression and clinicopathological significance of TIPE2 in cholangiocarcinoma. ( A ) Representative IHC staining of TIPE2 in cholangiocarcinoma tissues and adjacent normal tissues (× 200); LNM-: lymph node metastasis negative, LNM + : lymph node metastasis positive. ( B ) IHC sum scores were used to evaluate TIPE2 expression in cholangiocarcinoma tissues and adjacent normal tissues. ( C ) IHC sum scores were used to access TIPE2 expression in cholangiocarcinoma tissues with or without lymph node metastasis. ( D , E) TIPE2 IHC sum scores in different stages of cholangiocarcinoma. ( F – H ) ROC curves were established to evaluate the clinicopathological significance of TIPE2 in cholangiocarcinoma. ( F ) Tumor tissues v.s. normal tissues; ( G ) Tumor tissues without lymph node metastasis v.s. tumor tissues with lymph node metastasis; ( H ) Tumor tissues with different TNM stage. ( I ) Survival analysis according to TIPE2 expression in the cohort of patients with cholangiocarcinoma. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The sections were incubated with the blocking goat serum for 15 min and then immunostained with rabbit antibody against TIPE2 (dilution 1:300, BOSTER, China) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: TIPE2 serves as a favorable prognostic biomarker and suppresses cholangiocarcinoma progression by targeting RAC1-mediated integrin αvβ6 trafficking

doi: 10.1038/s41598-026-37540-9

Figure Lengend Snippet: TIPE2 suppresses the malignant behaviors of cholangiocarcinoma both in vitro and in vivo. ( A ) The expression of TIPE2 were detected after TIPE2 silencing or overexpression using RT-PCR. ( B ) CCK-8 assays were performed following TIPE2 silencing in RBE and QBC939 cells. ( C ) CCK-8 assays were performed following TIPE2 overexpression in RBE and QBC939 cells. ( D ) Trans-well migration and invasion assays were conducted after TIPE2 silencing in RBE cells. ( E ) Trans-well migration and invasion assays were conducted after TIPE2 silencing in QBC393 cells. ( F ) Trans-well migration and invasion assays were conducted after TIPE2 overexpression in RBE cells. ( G ) Trans-well migration and invasion assays were conducted after TIPE2 overexpression in QBC939 cells. ( H ) Tumor growth curves in the subcutaneous xenograft model for the TIPE2-OE group and the MOCK group. ( I ) Representative pictures of xenograft subcutaneous tumors in the TIPE2-OE group and the MOCK group. (bar = 1 cm). ( J ) Tumor volumes in the TIPE2 group and the MOCK group. ( K ) Tumor weights in different groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The sections were incubated with the blocking goat serum for 15 min and then immunostained with rabbit antibody against TIPE2 (dilution 1:300, BOSTER, China) at 4 °C overnight.

Techniques: In Vitro, In Vivo, Expressing, Over Expression, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Migration

Journal: Scientific Reports

Article Title: TIPE2 serves as a favorable prognostic biomarker and suppresses cholangiocarcinoma progression by targeting RAC1-mediated integrin αvβ6 trafficking

doi: 10.1038/s41598-026-37540-9

Figure Lengend Snippet: RAC1 was involved in the trafficking of integrin αvβ6. ( A ) Internalization assays were conducted in RBE and QBC939 cells after TIPE2 overexpression. ( B ) Recycling assays were conducted in RBE and QBC939 cells following TIPE2 overexpression. ( C ) Internalization assays were conducted in RBE and QBC939 cells that has been pre-treated with NSC23766. ( D ) Recycling assays were conducted in RBE and QBC939 cells that has been pre-treated with NSC23766. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The sections were incubated with the blocking goat serum for 15 min and then immunostained with rabbit antibody against TIPE2 (dilution 1:300, BOSTER, China) at 4 °C overnight.

Techniques: Over Expression

Journal: Scientific Reports

Article Title: TIPE2 serves as a favorable prognostic biomarker and suppresses cholangiocarcinoma progression by targeting RAC1-mediated integrin αvβ6 trafficking

doi: 10.1038/s41598-026-37540-9

Figure Lengend Snippet: TIPE2 targeted RAC1 and suppressed the trafficking of integrin αvβ6. ( A ) Total view of the interaction between the integrin β6 (cyan) and RAC1 (green). ( B ) Detailed view of the interaction between the integrin β6 (cyan) and RAC1 (green). ( C ) Total view of the interaction between the TIPE2 (rose red) and the integrin β6 (cyan). ( D ) Detailed view of the interaction between the TIPE2 (rose red) and the integrin β6 (cyan). ( E ) CCK-8 assays were carried out in cholangiocarcinoma cells transfected with either the TIPE2 overexpression plasmid or the Mutant plasmid. ( F ) Trans-well migration assays were carried out in cholangiocarcinoma cells transfected with either the TIPE2 overexpression plasmid or the Mutant plasmid. ( G ) Trans-well invasion assays were carried out in cholangiocarcinoma cells transfected with either a TIPE2 overexpression plasmid or a Mutant plasmid. ( H ) CCK-8 assays were carried out after TIPE2 overexpression in cholangiocarcinoma cells with or without NSC23766 pre-treatment. ( I ) Trans-well migration assays were carried out after TIPE2 overexpression in cholangiocarcinoma cells with or without NSC23766 pre-treatment. ( J ) Trans-well invasion assays were carried out after TIPE2 overexpression in cholangiocarcinoma cells with or without NSC23766 pre-treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The sections were incubated with the blocking goat serum for 15 min and then immunostained with rabbit antibody against TIPE2 (dilution 1:300, BOSTER, China) at 4 °C overnight.

Techniques: CCK-8 Assay, Transfection, Over Expression, Plasmid Preparation, Mutagenesis, Migration

Journal: Molecular Cell

Article Title: The Anti-inflammatory TIPE2 Is an Inhibitor of the Oncogenic Ras

doi: 10.1016/j.molcel.2012.01.006

Figure Lengend Snippet: (a) Interaction between endogenous RGL and TIPE2. Raw 264.7 cell lysates were immunoprecipitated (IP) with mouse anti-TIPE2 or control IgG, and subjected to SDS-PAGE and immunoblotting (IB). (b) Schematics of the RGL constructs used in this study. GEF, guanine nucleotide exchange factor; RID, Ras-interacting domain. (c) Interaction of TIPE2 with RGL Ras-interacting region. Lysates of 293T cells transiently transfected with RGL constructs and a TIPE2-Flag construct were immunoprecipitated with anti-Myc, and subjected to SDS-PAGE and IB. (d) TIPE2 prevents Ras from binding to RGL. Lysates of 293T cells transiently transfected with RGL-Myc (2 μg/10-cm plate) and increasing amounts of TIPE2-Flag constructs (5 μg to 10 μg/10-cm plate) were immunoprecipitated with anti-Myc, and subjected to SDS-PAGE and IB. (e) 293T cells were transiently transfected with TIPE2-Flag or pRK5 plasmids. 18 hours later, cell lysates were subjected to pull-down using GST-RalBP1 RBD. RalA from the pull-down and total RalA in the lysates were determined by IB. (f) TIPE2 Δ105–132 does not bind RGL. 293T cells were transielntly transfected with either TIPE2-Flag or TIPE2 Δ105–132 constructs, with or without RGL plasmid. 18 hours later, cell lysates were immunoprecipitated with anti-Myc antibody, and subjected to SDS-PAGE and IB. (g) Active and total RalA levels in wild type and Tipe2−/− bone marrow-derived murine macrophages were determined as in panel (e). Data shown are representative of 3 independent experiments.

Article Snippet: 40 μl of 50% proein G-sepharose beads was incubated for 1 hour at 4 °C, with one of the following antibodies: myc (1:1000, cell signaling), Flag (2 μg, Sigma), TIPE2 (1:500, Novus biological), and Sec5 (2 μg, ProteinTech) antibodies, or IgG isotype controls (BD biosciences).

Techniques: Immunoprecipitation, Control, SDS Page, Western Blot, Construct, Transfection, Binding Assay, Plasmid Preparation, Derivative Assay

Journal: Molecular Cell

Article Title: The Anti-inflammatory TIPE2 Is an Inhibitor of the Oncogenic Ras

doi: 10.1016/j.molcel.2012.01.006

Figure Lengend Snippet: (a) TIPE2 promotes cell death in 293T cells. Cells were transfected with the indicated amounts of TIPE2-Flag construct (TIPE2) or control GFP vector (GFP). After 24 hrs, cell death was assessed by trypan blue staining. Data shown are means ± S.E.M (n=3), and are representative of 5 independent experiments. ** p < 0.01; *** p < 0.001. (b) RGL rescues TIPE2-induced cell death. 293T cells were transfected with TIPE2-Flag together with RGL or RGL ΔN plasmids for 24 hrs, and cell death was assessed as in (a). Data shown are means ± S.E.M (n=5), and are representative of 5 independent experiments. ** p < 0.02. (c) TIPE2-induced death is mediated by the PDK1-AKT axis. 293T cells were transfected with TIPE2-Flag or TIPE2 Δ3105-132 plasmids, with or without activated RalA and RalB, wild type RalA and RalB, activated AKT, or myristoylated PDK1 plasmids for 24 hrs. Cell death was assessed as in (a). Data shown are means ± S.E.M (n=3). *** p< 0.001, ** p< 0.005. (d) AKT inhibition is responsible for TIPE2-induced cell death. 293T cells were transiently transfected with either GFP, TIPE2, dominant negative (DN) AKT, GFP plus TIPE2, or DN AKT plus TIPE2 plasmids. Cell death was assessed as in (a). Data shown are means ± S.E.M (n=5) of the cell death rates, and are pooled from 2 independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. (e) TIPE2 reduces phospho (P)-AKT (S473) levels. 293T cells were transfected with the indicated constructs for 24 hours, and protein levels were determined by IB. (f) TIPE2 decreases RGL interaction with PDK1. Lysates of 293T cells transiently transfected with RGL-Myc (2 μg/10-cm plate) and increasing amounts of TIPE2-Flag constructs (5 μg to 10 μg/10-cm plate) were immunoprecipitated with anti-Myc, and subjected to SDS-PAGE and IB. (g) Increased phosphorylation of AKT in Tipe2−/− splenocytes. Wild type and Tipe2−/− splenocytes were treated with LPS (200 ng/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and IB. Data shown are representative of three independent experiments. (h) Tipe2−/− splenocytes are resistant to serum deprivation-induced death. Tipe2−/− or wild type splenocytes were incubated in DMEM containing 10% (control) or 0.2% (low serum) FBS for 4 hrs, and cell death was assessed as in (a). Data shown are means ± S.E.M (n=3), and are representative of three independent experiments. * p < 0.05.

Article Snippet: 40 μl of 50% proein G-sepharose beads was incubated for 1 hour at 4 °C, with one of the following antibodies: myc (1:1000, cell signaling), Flag (2 μg, Sigma), TIPE2 (1:500, Novus biological), and Sec5 (2 μg, ProteinTech) antibodies, or IgG isotype controls (BD biosciences).

Techniques: Transfection, Construct, Control, Plasmid Preparation, Staining, Inhibition, Dominant Negative Mutation, Immunoprecipitation, SDS Page, Phospho-proteomics, Incubation

Journal: Molecular Cell

Article Title: The Anti-inflammatory TIPE2 Is an Inhibitor of the Oncogenic Ras

doi: 10.1016/j.molcel.2012.01.006

Figure Lengend Snippet: (a) Decreased rate of F-actin polymerization in TIPE2-expressing Raw 264.7 macrophages. Cells were transfected with either TIPE2 or empty plasmid (pRK5). One day later, the cells were treated with LPS (200 ng/ml) for the indicated times, and F-actin level was measured as described in Methods. The value of the TIPE2 group at time zero was set as 1. Results are means ± S.E.M, and were pooled from three independent experiments (n=12). * p < 0.05; ** p<0.01. (b) Increased polymerization of F-actin in Tipe2−/− splenocytes. Wild type and Tipe2−/− splenocytes were treated with LPS (200 ng/ml) for the indicated times and the F-actin level was measured. The value at time zero was set as 1. Results are means ± S.E.M, and were pooled from three independent experiments (n=6). * p < 0.05; ** p < 0.02. (c–d) Increased exocyst complex assembly in Tipe2−/− bone marrow-derived macrophages. Lysates of wild type and Tipe2−/− cells were immunoprecipitated with anti-Sec5 and subjected to SDS-PAGE and IB (c). Quantitation of the exocyst assembly was performed by densitometry (d). Results are means ± S.E.M of Sec6/Sec5 and Exo84/Sec5 ratios pooled from two independent experiments. The values of Sec6/Sec5 ratio for each group were set as 1. (e–f) Decreased exocyst complexes in TIPE2-expressing cells. Lysates from 293T cells transfected with increasing amounts of TIPE2 plasmid (0, 6, and 12 μg/10-cm plate) for 18 hours were immunoprecipitated and tested as in panel c (e). Quantitation of the exocyst assembly was performed by densitometry (f). Results are means ± S.E.M pooled from two independent experiments. The values of Sec6/Sec5 ratio for each group were set as 1. (g–h) Increased migration of Tipe2−/− macrophages. Confluent monolayers of wild type and Tipe2−/− bone marrow-derived macrophages were subjected to the wound-healing assay as described in Methods. Images were then taken immediately after applying the wound (time 0), and 3 and 6 hours later (g). Cells that migrated into the wounded areas were counted using the ImageJ software. Results are means ± S.E.M of 10 fields from each group, and are representative of three independent experiments. *p < 0.005.

Article Snippet: 40 μl of 50% proein G-sepharose beads was incubated for 1 hour at 4 °C, with one of the following antibodies: myc (1:1000, cell signaling), Flag (2 μg, Sigma), TIPE2 (1:500, Novus biological), and Sec5 (2 μg, ProteinTech) antibodies, or IgG isotype controls (BD biosciences).

Techniques: Expressing, Transfection, Plasmid Preparation, Derivative Assay, Immunoprecipitation, SDS Page, Quantitation Assay, Migration, Wound Healing Assay, Software

Journal: Molecular Cell

Article Title: The Anti-inflammatory TIPE2 Is an Inhibitor of the Oncogenic Ras

doi: 10.1016/j.molcel.2012.01.006

Figure Lengend Snippet: (a) TIPE2 inhibits the growth of Ras-transformed cells. Ras-transformed NIH3T3 cells that did or did not express TIPE2 were cultured in DMEM containing 0.5% FBS. Cells were fixed at 24 hr intervals and stained with methylene blue. Results are means ± S.E.M (n=5) of relative absorbance with the value at day one set as 1, and are representative of three independent experiments. ** p<0.001; *** p < 0.0001. (b) TIPE2 inhibits soft-agar colony formation of Ras-transformed cells. Ras-transformed NIH3T3 cells that did or did not express TIPE2 were cultured in duplicates in soft-agar plates as described in Methods, and colonies were counted in 20 random fields for each culture under 40X magnification. Results are means ± S.E.M (n=20) and are representative of three independent experiments. * p < 0.0001. (c) TIPE2 delays tumorigenesis in nude mice. Ras-transformed NIH3T3 cells that did or did not express TIPE2 were injected subcutaneously into the rear flanks of nude mice (2×106 cells/injection, n=3), and tumor formation was monitored daily. All sites injected with Ras-transformed NIH3T3 cells eventually developed tumors, whereas control NIH3T3 or NIH3T3-TIPE2 cell lines did not give rise to tumors during the course of this study. Data shown are percent of tumors formed, and are representative of two independent experiments. The differences between the two groups are statistically significant (p = 0.014). (d–e) Loss of TIPE2 protein expression in Ras-transformed NIH3T3 tumors. Tumors from two separate experiments were excised at day 14 or day 22. Expression of TIPE2-Flag protein (d) and mRNA (e) was examined by SDS-PAGE and IB, and qRT-PCR, respectively. Cell lines used in these assays were Ras-transformed NIH3T3 cells that did (TIPE2) or did not (control) express TIPE2-Flag. qRT-PCR data are shown as means ± S.E.M (n=3) with the value of the control group set as 1. (f) Ubiquitination of TIPE2. 293T cells were transfected with either TIPE2-Flag, or p50-Flag as a positive control. The cells were treated with MG132 (10μM), the lysates were immunoprecipitated with an antibody against Flag, and the ubiquitination status was examined as we previously described (Carmody et al., 2007). (g) TIPE2 protein is degraded in Ras-transformed NIH3T3 tumors. Reconstituted cells from Ras3T3-TIPE2 tumors were treated with MG132 for 24 hrs and TIPE2 protein levels were examined by Western blot and compared to untreated pre-injected cells. (h–j) Loss of, or decreased, TIPE2 expression in human hepatocellular carcinoma. TIPE2 expression in tumor tissue (h) and control hepatic tissue adjacent to the tumor (i) from the same patient was determined by immunohistochemistry as described in Methods. TIPE2-positive cells are shown in brown. Original magnification, x400. Quantitation of the TIPE2 signal for 116 patients was performed as described in Methods (j). The differences between the two groups are statistically significant (p < 0.001).

Article Snippet: 40 μl of 50% proein G-sepharose beads was incubated for 1 hour at 4 °C, with one of the following antibodies: myc (1:1000, cell signaling), Flag (2 μg, Sigma), TIPE2 (1:500, Novus biological), and Sec5 (2 μg, ProteinTech) antibodies, or IgG isotype controls (BD biosciences).

Techniques: Transformation Assay, Cell Culture, Staining, Injection, Control, Expressing, SDS Page, Quantitative RT-PCR, Ubiquitin Proteomics, Transfection, Positive Control, Immunoprecipitation, Western Blot, Immunohistochemistry, Quantitation Assay

Journal: Cell & Bioscience

Article Title: TIPE2 acts as a biomarker for tumor aggressiveness and suppresses cell invasiveness in papillary thyroid cancer (PTC)

doi: 10.1186/s13578-018-0247-x

Figure Lengend Snippet: Correlations between TIPE2 expression and clinicopathological characteristics in PTC

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against TIPE2 (1:300; Abcam, UK), or anti-β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 h at room temperature.

Techniques: Expressing

Journal: Cell & Bioscience

Article Title: TIPE2 acts as a biomarker for tumor aggressiveness and suppresses cell invasiveness in papillary thyroid cancer (PTC)

doi: 10.1186/s13578-018-0247-x

Figure Lengend Snippet: TIPE2 overexpression markedly suppressed the viability and proliferation of PTC cells. A-B. Real-time PCR ( a ) and western-blot ( b ) showed that TIPE2 expression was significantly upregulated after transfection of the PRK5-TIPE2 plasmid in TPC-1 and B-CPAP cells. c , d After TIPE2 overexpression, CCK8 assays were conducted to evaluate the viability of TPC-1 ( c ) and B-CPAP cells ( d ). e , f MTT assays were conducted to evaluate the proliferation of TPC-1 ( e ) and B-CPAP cells ( f ) after TIPE2 overexpression. Data represent the mean ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against TIPE2 (1:300; Abcam, UK), or anti-β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 h at room temperature.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: TIPE2 acts as a biomarker for tumor aggressiveness and suppresses cell invasiveness in papillary thyroid cancer (PTC)

doi: 10.1186/s13578-018-0247-x

Figure Lengend Snippet: TIPE2 notably suppressed the migration and invasion of PTC cells. a TPC-1 cells transfected with the TIPE2 overexpression plasmid were used for Transwell migration and invasion assays. b Statistic results were shown by counting five fields of TPC-1 cells in the lower chambers (×200 magnification). c B-CPAP cells transfected with the TIPE2 overexpression plasmid were used for Transwell migration and invasion assays. d Statistic results were shown by counting five fields of B-CPAP cells in the lower chambers (×200 magnification). Data represent the mean ± SD of three independent experiments. ** P < 0.01; *** P < 0.001

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against TIPE2 (1:300; Abcam, UK), or anti-β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 h at room temperature.

Techniques: Migration, Transfection, Over Expression, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: TIPE2 acts as a biomarker for tumor aggressiveness and suppresses cell invasiveness in papillary thyroid cancer (PTC)

doi: 10.1186/s13578-018-0247-x

Figure Lengend Snippet: TIPE2 suppressed the invasiveness of PTC cells via inhibiting Rac1. a IHC results (×200 magnification) of Rac1 expression in PTC tissues with or without infiltration. b IHC sum scores were used to compare TIPE2 expression in PTC tissues with or without infiltration. c The invasiveness of TPC-1 and B-CPAP cells were measured by Transwell invasion assays after transfection of specific Rac1 siRNA. d After treatment of NSC23766, Transwell invasion assays were used to evaluate the invasiveness of TPC-1 and B-CPAP cells. e TPC-1 and B-CPAP cells co-transfected with Rac1-specific siRNA and TIPE2 overexpressing plasmid were used for Transwell invasion assays. f TPC-1 and B-CPAP cells that transfected with Mock, wild type TIPE2 and mutant TIPE2 plasmids were used for Transwell invasion assays. Data are shown as the mean ± SD, and the results shown are representative of 3 independent experiments. Five fields of cells in the lower compartment were counted in Transwell assays (200 × magnification). ** P < 0.01

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against TIPE2 (1:300; Abcam, UK), or anti-β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 h at room temperature.

Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Cell & Bioscience

Article Title: TIPE2 acts as a biomarker for tumor aggressiveness and suppresses cell invasiveness in papillary thyroid cancer (PTC)

doi: 10.1186/s13578-018-0247-x

Figure Lengend Snippet: TIPE2 suppressed uPA and MMP9 expression via inhibiting Rac1 in PTC cells. a The expression of uPA and MMP9 at mRNA level were detected by real-time PCR after transfection with Mock, wild type TIPE2 and mutant TIPE2 plasmids in PTC cells. b The protein level of uPA and MMP9 were detected by ELISA in PTC cells after transfected with Mock, wild type TIPE2 and mutant TIPE2 plasmids. c The expression of uPA and MMP9 at mRNA level were detected by real-time PCR in TPC-1 and B-CPAP cells after Rac1 silencing. d The protein level of uPA and MMP9 were detected by ELISA in TPC-1 and B-CPAP cells after Rac1 silencing

Article Snippet: Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against TIPE2 (1:300; Abcam, UK), or anti-β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: TNFAIP8 is a novel phosphoinositide-binding inhibitory regulator of Rho GTPases that promotes cancer cell migration

doi: 10.1101/2021.03.26.437243

Figure Lengend Snippet: ( a ) Cartoon presentation of murine TNFAIP8 C165S mutant (PDB accession 5JXD) in complex with a phospholipid shown by ball model. Helices are rainbow colored from α 0 (in blue) to α 6 (in red). The surface of the centrally located hydrophobic cavity is shown in grey. ( b ) Comparison of protein topography of murine TNFAIP8 C165S, human TIPE2, human TIPE3, and human RhoGDI1 crystal structures by CASTp 3.0. ( c ) For the 31 amino acids with hydrophobic side chains that line the cavity of murine TNFAIP8 C165S, identical (represented by circles) or similar amino acids in the corresponding positions are indicated. ( d , e ) Representation of mutated TNFAIP8 residues. Four conserved residues L44 ( α 1), L65 ( α 2), L99 ( α 3), and L158 ( α 5) (highlighted in magenta) out of the wall residues comprising the pocket (highlighted in yellow) were mutated to Ws to induce steric blockade in the cavity ( d ). Two positively charged residues of α 0 helix K17 and K18 (highlighted in orange) were mutated to Qs for the 2Q mutant, and two additional residues K22 and K26 (highlighted in orange) were replaced by Qs to construct the 4Q mutant ( e ). Three positively charged amino acids H76, R77, and K93 (highlighted in blue) positioned at the opening of the cavity were mutated to Qs for the Entrance mutation ( e ). Residues are shown in stick models and cavity surface is colored grey. ( f , g ) Binding of 6His-SUMO tagged TNFAIP8, 4Q, Entrance, and L158W mutant proteins to the indicated lipids spotted on strips by protein-lipid overlay assay. Immunoblot was performed using antibody against 6His-tag. Signal from wild-type TNFAIP8 was set as 1. Data represent three independent experiments with similar results ( f ) or are mean ± s.e.m. of three independent experiments ( g ). P values are from two-sided unpaired Student’s t -test ( g ).

Article Snippet: Anti-TNFAIP8 and TIPE2 antibodies were purchased from Proteintech.

Techniques: Mutagenesis, Comparison, Construct, Binding Assay, Protein-lipid Overlay Assay (PLOA), Western Blot

Journal: Neurochemical research

Article Title: Exogenous TIPE2 Inhibit TAK1 to Improve Inflammation and Neuropathic Pain Induced by Sciatic Nerve Injury Through Inactivating NF-κB and JNK.

doi: 10.1007/s11064-022-03671-4

Figure Lengend Snippet: Fig. 7 A possible process of TIPE2 inhibition of activation of TAK1 signaling. Binding between aa101-104 region on TIPE2 and aa 200-291 region of the internal kinase domain of TAK1 blocked the formation of TAK1-TAK1-binding protein (TAB) 1-TAB2 complex, further to interfer TAK1 activa- tion. Therefore, the negative regulation by TIPE2 of TAK1 activation may lead to inhibi- tion of TAK1-NF-κB mediated inflammatory responses

Article Snippet: After blocking by 5% non-fat milk, the membranes were incubated with primary antibodies against p-IκBα (1:10000, ab133462, Abcam), IκBα (1:800, ab95338, Abcam), p-JNK1(1:1200, ab47337, Abcam), p-JNK2 (1:1000, P02706, Boster), JNK1 (1:2500, ab199380, Abcam), JNK2 (1:1500, 51153-1-AP, Proteintech), β-actin (1:2000, 20536-1-AP, Proteintech), TIPE2 (1:1000, orb158628, Biorbyt), TAK1 (1:800, orb256677, Biorbyt) at 4 °C overnight.

Techniques: Inhibition, Activation Assay, Binding Assay