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: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 1 TLR5 expression in the myocardium after AB-induced pressure overload. Western

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques: Expressing, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 2 the expression of TLR5 in cultured neonatal rat cardiomyocytes, H9c2

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques: Expressing, Cell Culture

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 3 The effects of TLR5 on cardiac dysfunction. Statistical analysis of the HW/BW ratio,

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques:

Journal: Biochimica et biophysica acta

Article Title: Toll-like receptor 5 deficiency attenuates interstitial cardiac fibrosis and dysfunction induced by pressure overload by inhibiting inflammation and the endothelial-mesenchymal transition.

doi: 10.1016/j.bbadis.2015.08.013

Figure Lengend Snippet: Figure 4 TLR5 deficiency attenuates the fibrotic response induced by pressure overload.

Article Snippet: Before TGF-β1 treatment, the HUVEC-12 cells were transfected with a plasmid over-expressing TLR5 (Ad-TLR5, Addgene, 13019) using FuGENE®HD Transfection Reagent (Promega, E2311).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Antagonists of Anaphase-promoting Complex (APC)-2-Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 Interaction Are Novel Regulators of Cell Growth and Apoptosis

doi: 10.1074/jbc.m111.222398

Figure Lengend Snippet: FIGURE 3. CARP-1 interacts with Cdc20 and Cdh1. A, protein complexes derived from wild-type MDA-MB-468 HBC cells (upper blot) or COS-7 cells transfected with noted plasmids (lower blot) were subjected to IP using indicated antibodies, followed by WB of the immunoprecipitates or respective cell lysates as in Fig. 1. Cdh1-interacting epitope of CARP-1 is distinct from its APC-2-binding epitope. COS-7 cells were transfected with plasmids encoding myc-His-tagged wild-type (WT) CARP-1, its 896–978 mutant (B), or different CARP-1 mutants (C) in combination with plasmid expressing GST-Cdh1. The cell lysates were subjected to IP-WB using indicated antibodies as in Fig. 1. D, apoptosis signaling regulates CARP-1 binding with APC-2. Protein complexes derived from wild-type untreated (NT), serum-starved (ST), or nocodazole-treated (NZ) HBC cells were subjected to IP using indicated antibodies. Immunoprecipitates or cell lysates were analyzed by WB with noted antibodies essentially as in Fig 1. Presence of the endogenous or the transfected proteins in A–D are indicated by an arrowhead on the left side of each blot except that the GST-tagged Cdc20 and Cdh1 are indicated in the respective lane of the lower blot of A. Approximate location of various molecular mass markers is indicated on the right side of each blot. kDa, kilodalton.

Article Snippet: The plasmid pCMV-SPORT6 having human NEMO (IKK ) or Deleted in breast cancer (Dbc)-1 (17) cDNAs were purchased from ATCC (Manassas, VA), and the plasmids having human APC-2, Cdc20, or Cdh1 cDNAs (18) were obtained from Addgene (Cambridge, MA).

Techniques: Derivative Assay, Transfection, Binding Assay, Mutagenesis, Plasmid Preparation, Expressing

Journal: Journal of Biological Chemistry

Article Title: Antagonists of Anaphase-promoting Complex (APC)-2-Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 Interaction Are Novel Regulators of Cell Growth and Apoptosis

doi: 10.1074/jbc.m111.222398

Figure Lengend Snippet: FIGURE 7. CFM-4 suppresses cell growth in part by elevating CARP-1 and diminishing cyclin B1 levels. A, cells were treated with the indicated agents for noted times, labeled with propidium iodide, and sorted by flow cytometry. Histogram and table below represent cell numbers in various phases of cell cycle. B–D, indicated cells were either untreated (), treated with DMSO (Control (DMSO)), or treated () with noted time and dose of respective agents. The cell lysates (50 g/lane) in B–D were analyzed by WB for levels of CARP-1, cyclin B1, Cdh1, APC-2, CDKIs p21WAF1/CIP1, p27KIP1 and actin proteins as under “Experimental Procedures.”

Article Snippet: The plasmid pCMV-SPORT6 having human NEMO (IKK ) or Deleted in breast cancer (Dbc)-1 (17) cDNAs were purchased from ATCC (Manassas, VA), and the plasmids having human APC-2, Cdc20, or Cdh1 cDNAs (18) were obtained from Addgene (Cambridge, MA).

Techniques: Labeling, Flow Cytometry, Control

Journal: Journal of Biological Chemistry

Article Title: Antagonists of Anaphase-promoting Complex (APC)-2-Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 Interaction Are Novel Regulators of Cell Growth and Apoptosis

doi: 10.1074/jbc.m111.222398

Figure Lengend Snippet: FIGURE 8. CARP-1 is required for cell growth inhibition by CFM-4, and cyclin B1 loss in the presence of CFM-4 is accomplished independent of UPP. Knockdown of CARP-1 or Cdh1 blocks CFM-4 effects. Cells were transfected with 100 nM each of the scrambled CARP-1 siRNAs (A and B) or Cdh1 siRNAs (C and D) for 72 h and were then either untreated or treated with CFM-4 for noted time and dose. Cell lysates were subjected to WB as in Fig. 7B (A and C) or subjected to MTT assay for determination of their viabilities (B and D). Columns in B and D represent means of three independent experiments; bars, S.E. * and #, p 0.01 relative to CFM-4-treated, scrambled siRNA-transfected cells. E, CFM-4 targets cyclin B1 independent of UPP. The cells were either treated with DMSO (control) or with noted dose and time of indicated agents. Cell lysates were then analyzed by WB as in A. Presence of various proteins is indicated by an arrowhead on the left side of each blot.

Article Snippet: The plasmid pCMV-SPORT6 having human NEMO (IKK ) or Deleted in breast cancer (Dbc)-1 (17) cDNAs were purchased from ATCC (Manassas, VA), and the plasmids having human APC-2, Cdc20, or Cdh1 cDNAs (18) were obtained from Addgene (Cambridge, MA).

Techniques: Inhibition, Knockdown, Transfection, MTT Assay, Control

Journal: Cell

Article Title: All-optical electrophysiology reveals the role of lateral inhibition in sensory processing in cortical layer 1

doi: 10.1016/j.cell.2020.01.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: i-Optopatch constructs include: LZF1826 pAAV_hSyn-DiO-SomArchon-eGFP-P2A-stGTACR2 (Addgene #135412) LZF1827 pAAV_hSyn-Flp-off-Cre-on-stGtACR2-CFP (Addgene #135413) pAAV_hSyn1-SIO-stGtACR2-FusionRed was a gift from Ofer Yizhar (Addgene plasmid # 105677). stGtACR2 cDNA segments were generated from the template of pAAV_hSyn1-SIO-stGtACR2-FusionRed (Addgene #105677).

Techniques: Virus, Plasmid Preparation, Recombinant, Transgenic Assay, Software, Microscopy