Journal: International journal of oncology

Article Title: TIP60 governs the auto‑ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex.

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: Figure 1. TIP60 and ubiquitin co‑transfection induces the downregulation of UHRF1. Cells were co‑transfected with either TIP60‑eGFP (green) and RFP‑Ubiquitin (red) or eGFP and RFP‑Ubiquitin. Immunostaining of UHRF1 in HeLa cells without (A) or with treatment by MG‑132 (B). Cells were fixed following transfection and labeled with anti‑UHRF1 antibody. Endogenous UHRF1 protein was labeled with Alexa 647‑labeled secondary antibody before visualization with confocal microscopy. Scale bar, 10 µm. (C and D) Represent mean fluorescence intensities levels of UHRF1 in the different samples. Values are the mean ± SEM for three independent experiments; *P<0.05; ****P<0.0001 (vs. control group), determined by one‑way ANOVA with Tukey's post hoc test. UHRF1, ubiquitin‑like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa.

Article Snippet: Other antibodies used included rabbit polyclonal anti‐HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti‐DNMT1 (1:5,000; cat. no. PTG‐MAB0079, ProteoGenix), mouse monoclonal anti‐ubiquitin (1:500; cat. no. 05‐944, Sigma‐Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002‐1‐Ig, Proteintech Group, Inc.; and cat. no. A‐11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti‐GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti‐GFP (1:1,000; cat. no. 66002‐1‐Ig, Proteintech Group, Inc.), mouse mono‐ clonal anti‐p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti‐caspase‐3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti‐BCL2 (1:1,000; cat. no. 05‐826, Merck KGaA), mouse monoclonal anti‐poly(ADP‐ribose) polymerase (PARP; 1:1,000; cat. no. 51‐6639GR, BD Biosciences) and rabbit polyclonal anti‐BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Ubiquitin Proteomics, Immunostaining, Transfection, Labeling, Confocal Microscopy, Fluorescence, Control

Journal: International journal of oncology

Article Title: TIP60 governs the auto‑ubiquitination of UHRF1 through USP7 dissociation from the UHRF1/USP7 complex.

doi: 10.3892/ijo.2021.5269

Figure Lengend Snippet: Figure 3. TIP60 induces auto‑ubiquitination of UHRF1 in HeLa cells. Cells stably expressing either UHRF1 WT or UHRF1 C724A‑H741A mutant were transfected with either TIP60-eGFP WT or TIP60ΔMYST‑eGFP mutant. All samples were treated with 10 µM of MG‑132, 8 h before harvesting the cells. Whole cell lysates and immunoprecipitated samples were analyzed by SDS‑PAGE and then immunoblotted with anti‑GFP and anti‑Ubiquitin antibodies. Inputs and IP gels were processed in parallel under similar conditions. UHRF1, ubiquitin‑like, containing PHD and RING finger domains 1; TIP60, Tat interactive protein, 60 kDa.

Article Snippet: Other antibodies used included rabbit polyclonal anti‐HAUSP/USP7 (1:5,000; cat. no. ab4080, Abcam), mouse monoclonal anti‐DNMT1 (1:5,000; cat. no. PTG‐MAB0079, ProteoGenix), mouse monoclonal anti‐ubiquitin (1:500; cat. no. 05‐944, Sigma‐Aldrich; Merck KGaA), mouse monoclonal eGFP (1:1,000; cat. no. 66,002‐1‐Ig, Proteintech Group, Inc.; and cat. no. A‐11120, Thermo Fisher Scientific, Inc.), mouse monoclonal anti‐GAPDH (1:5,000; cat. no. MAB374, Merck KGaA), mouse monoclonal anti‐GFP (1:1,000; cat. no. 66002‐1‐Ig, Proteintech Group, Inc.), mouse mono‐ clonal anti‐p73 (1:500; cat. no. 558785, BD Biosciences), rabbit polyclonal anti‐caspase‐3 (1:1,000; cat. no. 9661, Cell Signaling Technology, Inc.), mouse monoclonal anti‐BCL2 (1:1,000; cat. no. 05‐826, Merck KGaA), mouse monoclonal anti‐poly(ADP‐ribose) polymerase (PARP; 1:1,000; cat. no. 51‐6639GR, BD Biosciences) and rabbit polyclonal anti‐BAX (1:1,000; cat. no. AB2930, Merck KGaA).

Techniques: Stable Transfection, Expressing, Mutagenesis, Transfection, Immunoprecipitation

Journal: Cell

Article Title: LECT2, a Ligand for Tie1, Plays a Crucial Role in Liver Fibrogenesis.

doi: 10.1016/j.cell.2019.07.021

Figure Lengend Snippet: Figure 4. Direct Interactions between LECT2 and Tie1 (A) Schematic illustration of Tie1 and LECT2 constructs. (B) Exogenous Myc-tagged LECT2 and Flag-tagged Tie1 were immunoprecipitated. (C) Endogenous LECT2 and Tie1 were immunoprecipitated.

Article Snippet: The following primary antibodies were used for co-immunoprecipitation and western blotting: Anti-Flag antibody (1:1000, SigmaAldrich, F1804, RRID:AB_262044), anti-MYC antibody (1:1000, Proteintech, 66004-1-Ig), anti-His antibody (1:1000, proteintech, 66005-1-Ig, RRID:AB_11232599), anti-LECT2 antibody (1:1000, Abcam, ab196015), anti-Tie1 antibody (1:500, Invitrogen, PA527903, RRID:AB_2545379), anti-Tie1Phospho-Tyr1117 antibody (1:500, Abcam, AB12535), anti-Tie2 antibody (1 mg/ml, R&D, AF313, RRID:AB_355295), anti-Tie2-Phospho-Y992 antibody (0.5 mg/ml, R&D, AF2720, RRID:AB_442172), anti-p38 antibody (1:1000, Abcam, ab31828), anti-PhosphoY182-p38 antibody (1:2000, Abcam, ab47363) anti-Met antibody (1:1000, Cell Signaling Technology, 8198, RRID:AB_10858224), anti-Met-Phospho-Tyr1234/1235 antibody (1:1000, Cell Signaling Technology, 3077, RRID:AB_2315156), anti-VEGFR2 antibody (1:1000, Cell Signaling Technology, 2479, RRID:AB_2212507), anti-VEGFR2-PhosphoTyr1175 antibody (1:1000, Cell Signaling Technology, 2478, RRID:AB_331377), anti-VE-cadherin (1:2000, Abcam, ab33168), and anti-a-Tubulin antibody (1:1000, Beijing Ray Antibody Biotech, RM2007).

Techniques: Construct, Immunoprecipitation

Journal: Cell death & disease

Article Title: UHMK1 aids colorectal cancer cell proliferation and chemoresistance through augmenting IL-6/STAT3 signaling.

doi: 10.1038/s41419-022-04877-8

Figure Lengend Snippet: Fig. 3 The effects of UHMK1 upregulation on CRC cell proliferation, tumorigenicity and oxaliplatin resistance. A Western blot results of UHMK1 overexpression in DLD-1 and SW620 cells. Anti-UHMK1 (left) and anti-FLAG (right) antibodies were used. B The growth rates of DLD-1 and SW620 cells with or without UHMK1 overexpression were monitored by CCK-8 assay. C EdU staining was used to detect cell proliferation in DLD-1 cells with UHMK1 stable overexpression and control cells. The number of positive EdU cells was recorded. Scale bar: 100 μm. D The image of tumors removed from nude mice implanted with DLD-1/UHMK1-2 cells (n = 8) and control RKO cells (n = 8) for 3 weeks. Tumor weight and tumor volume were analyzed compared to control group. E Colony formation assays in UHMK1 stably overexpressed DLD-1 cells and control cells with or without oxaliplatin treatment (100 μM). F FACS analysis of apoptotic DLD-1 cells with or without oxaliplatin treatment (300 μM). UHMK1 stable expression cells had an anti-apoptosis effect. *p < 0.05, **P < 0.01.

Article Snippet: The primary antibodies are as follows: UHMK1 (#11624-1-AP), GAPDH (#60004-1-Ig), c-MYC (#10828-1-AP) and FLAG-like tag (#66008-3-Ig) were obtained from Proteintech, Wuhan, China.

Techniques: Western Blot, Over Expression, CCK-8 Assay, Staining, Control, Stable Transfection, Expressing