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