human trim33  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Identification of RSK substrates using an analog-sensitive kinase approach

doi: 10.1016/j.jbc.2024.105739

Figure Lengend Snippet: TRIM33 is phosphorylated by RSK at S1119. A , HEK293 cells were transfected with HA-tagged TRIM33, serum-starved overnight, and stimulated for 10 or 20 min with fetal bovine serum (FBS; 10%), EGF (25 ng/ml), insulin (100 nM), or PMA (50 ng/ml). Immunoprecipitated TRIM33 was then assayed for phosphorylation with a phospho-motif antibody that recognizes the RxxS∗/T∗ consensus motif. B , same as in ( A ), except cells were pretreated with PD184352 (10 μM) or BI-D1870 (10 μM) for 30 min prior to PMA stimulation. C , as in ( A ), except cells were transfected with HA-tagged TRIM33 WT or the S1119A unphosphorylable mutant. D , detection of phospho-RxxS∗/T∗ in TRIM33-WT and -S1119A in WT and RSK-DKO HeLa cells. Cells transfected with the HA-tagged TRIM33 constructs were stimulated for 30 min with PMA prior to HA immunoprecipitation. PMA, phorbol 12-myristate 13-acetate; RSK, ribosomal S6 kinase.

Article Snippet: The original plasmid encoding human TRIM33 (pCS2-FLAG-hEcto/Tif1g) was obtained from Dr Stefano Piccolo through Addgene (#20902).

Techniques: Transfection, Immunoprecipitation, Phospho-proteomics, Mutagenesis, Construct

Journal: Rheumatology (Oxford, England)

Article Title: ELISA, protein immunoprecipitation and line blot assays for anti-TIF1-gamma autoantibody detection in cancer-associated dermatomyositis

doi: 10.1093/rheumatology/keac288

Figure Lengend Snippet: Demographic data of anti-TIF1-gamma-positive and -negative patients detected by in-house ELISA, IP and LB assays

Article Snippet: An in-house ELISA was performed using recombinant full-length human TIF1-gamma protein (OriGene, Rockville, MD, USA) which was diluted in carbonate buffer at 0.25 μg/ml, pH 9.6, and coated on 384-well high binding plates (Corning, Sigma-Arldrich, Burlington, MA, USA) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Rheumatology (Oxford, England)

Article Title: ELISA, protein immunoprecipitation and line blot assays for anti-TIF1-gamma autoantibody detection in cancer-associated dermatomyositis

doi: 10.1093/rheumatology/keac288

Figure Lengend Snippet: The distribution of anti-TIF1-gamma levels in the LB and ELISAs among HC and DM patients Panels ( A ), ( B ) and ( C ) demonstrate anti-TIF1-gamma autoantibody levels from a line blot (LB) assay (A) and in-house ELISA (B) among healthy controls (HC) and patients with DM, and anti-TIF1-gamma autoantibody levels from a commercial ELISA (C) among patients with DM. Each data point represents one individual and horizontal lines (black) indicate the mean values. DU: density units.

Article Snippet: An in-house ELISA was performed using recombinant full-length human TIF1-gamma protein (OriGene, Rockville, MD, USA) which was diluted in carbonate buffer at 0.25 μg/ml, pH 9.6, and coated on 384-well high binding plates (Corning, Sigma-Arldrich, Burlington, MA, USA) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of TRIM33 Promotes Survival and Epithelial–Mesenchymal Transition in Gastric Cancer

doi: 10.1177/15330338221114505

Figure Lengend Snippet: The expression of TRIM33 in pan-cancer, and association between TRIM33 expression and the survival of patients with GC. (A) TRIM33 expression in 31 tumors from TCGA database. Red bars represent tumor samples, blue bars represent normal tissue samples. (B) OS, FPS, and PPS curves in patients with GC. (C) Survival curves of different pTNM stages in patients with GC. (D) Survival curves of different pT stages in patients with GC. * P < .05, ** P < .01, *** P < .001 versus normal tissue samples.

Article Snippet: The rabbit antibody against human TRIM33 (Cat No. 55374-1-AP) was purchased from ProteinTech; rabbit antibodies against human phospho-Smad2 (p-Smad2; #18338), Smad2 (#5339), Smad3 (#9523), Smad4 (#46535), vimentin (#5741), N-Cadherin (#13116), E-Cadherin (#3195), and β-actin (#5125), as well as a horseradish peroxidase-linked goat anti-rabbit antibody (#7074), were purchased from Cell Signaling Technology.

Techniques: Expressing

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of TRIM33 Promotes Survival and Epithelial–Mesenchymal Transition in Gastric Cancer

doi: 10.1177/15330338221114505

Figure Lengend Snippet: Knockdown of TRIM33 promoted the proliferation of BGC-823 and SGC-7901 cells. (A&B) Western blot analysis of TRIM33 expression in GES, BGC-823, MGC-803, and SGC-7901 cell lines. (C&D) Western blot analysis of TRIM33 protein expression in cells transfected with si-TRIM33 encoding plasmids. (E) CCK8 assays were used to measure BGC-823 and SGC-7901 cell viability in the control and si-TRIM33-transfected group; the viability of the si-TRIM33 transfection groups was enhanced. (F&G) EdU assays were used to evaluate proliferation in the control and si-TRIM33 transfection groups in BGC-823 cells; the percentage of EdU-positive cells in the si-TRIM33 group was significantly greater than in the control group. * P < .05, ** P < .01; ns, non-significant.

Article Snippet: The rabbit antibody against human TRIM33 (Cat No. 55374-1-AP) was purchased from ProteinTech; rabbit antibodies against human phospho-Smad2 (p-Smad2; #18338), Smad2 (#5339), Smad3 (#9523), Smad4 (#46535), vimentin (#5741), N-Cadherin (#13116), E-Cadherin (#3195), and β-actin (#5125), as well as a horseradish peroxidase-linked goat anti-rabbit antibody (#7074), were purchased from Cell Signaling Technology.

Techniques: Knockdown, Western Blot, Expressing, Transfection, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of TRIM33 Promotes Survival and Epithelial–Mesenchymal Transition in Gastric Cancer

doi: 10.1177/15330338221114505

Figure Lengend Snippet: Downregulation of TRIM33 enhanced colony formation and migratory ability. (A&B) Downregulation of TRIM33 promoted colony formation in BGC-823 and SGC-7901 cells. (C&D) After 24 h, the number of cells passing through the Transwell membrane was higher in the si-TRIM33 group than in the control group (×100 magnification, corresponding to a scale of 200 μm). (E&F) After 12, 24, 48, 72, 96, 120, and 144 h, the width of the residual scratch was narrower in the si-TRIM33 group than in the control group (×100 magnification, corresponding to a scale of 200 μm). * P < .05, ** P < .01.

Article Snippet: The rabbit antibody against human TRIM33 (Cat No. 55374-1-AP) was purchased from ProteinTech; rabbit antibodies against human phospho-Smad2 (p-Smad2; #18338), Smad2 (#5339), Smad3 (#9523), Smad4 (#46535), vimentin (#5741), N-Cadherin (#13116), E-Cadherin (#3195), and β-actin (#5125), as well as a horseradish peroxidase-linked goat anti-rabbit antibody (#7074), were purchased from Cell Signaling Technology.

Techniques: Membrane, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of TRIM33 Promotes Survival and Epithelial–Mesenchymal Transition in Gastric Cancer

doi: 10.1177/15330338221114505

Figure Lengend Snippet: Knockdown of TRIM33 upregulated TGF-β expression. (A) ELISA analysis of TGF-β expression in the si-TRIM33 and control groups identified that knockdown of TRIM33 upregulated TGF-β expression, suggesting activation of the TGF-β signaling pathway. (B&C) Western blot analysis of the expression of proteins in the TGF-β signaling pathway in control, si-NC, and si-TRIM33 cells. Expression of p-Smad2 (Ser465/467), Smad2, Smad3, Smad4, vimentin, and N-Cadherin was upregulated, and E-Cadherin expression was downregulated, in si-TRIM33 cells. * P < .05, ** P < .01.

Article Snippet: The rabbit antibody against human TRIM33 (Cat No. 55374-1-AP) was purchased from ProteinTech; rabbit antibodies against human phospho-Smad2 (p-Smad2; #18338), Smad2 (#5339), Smad3 (#9523), Smad4 (#46535), vimentin (#5741), N-Cadherin (#13116), E-Cadherin (#3195), and β-actin (#5125), as well as a horseradish peroxidase-linked goat anti-rabbit antibody (#7074), were purchased from Cell Signaling Technology.

Techniques: Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Control, Activation Assay, Western Blot

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of TRIM33 Promotes Survival and Epithelial–Mesenchymal Transition in Gastric Cancer

doi: 10.1177/15330338221114505

Figure Lengend Snippet: Knockdown of TRIM33 promoted GC growth in vivo. (A) The xenograft model of TRIM33-knockdown revealed that tumors were significantly larger in si-TRIM33 animals than in controls. (B&C) Immunohistochemical analysis identified that the protein levels of TRIM33, p-Smad2 (Ser465/467), Smad2, Smad3, Smad4, vimentin, and N-Cadherin were increased, and levels of E-Cadherin were decreased, in xenograft tumors from the si-TRIM33 group (×100 and ×400 magnification in left- and right-hand side images, corresponding to a scale of 200 and 50 μm, respectively).

Article Snippet: The rabbit antibody against human TRIM33 (Cat No. 55374-1-AP) was purchased from ProteinTech; rabbit antibodies against human phospho-Smad2 (p-Smad2; #18338), Smad2 (#5339), Smad3 (#9523), Smad4 (#46535), vimentin (#5741), N-Cadherin (#13116), E-Cadherin (#3195), and β-actin (#5125), as well as a horseradish peroxidase-linked goat anti-rabbit antibody (#7074), were purchased from Cell Signaling Technology.

Techniques: Knockdown, In Vivo, Immunohistochemical staining