protein graph  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Reciprocal antagonism of PIN1-APC/C CDH1 governs mitotic protein stability and cell cycle entry

doi: 10.1038/s41467-024-47427-w

Figure Lengend Snippet: a 293T cells were transfected with indicated constructs for 36 hrs. Input is 5% of the total lysates used in IP. b In vitro kinase assay showing that CDK4 phosphorylates CDH1 at Ser163. c FACS analysis of APC/C-degron reporter levels in MCF-7 cells expressing either WT CDH1, S163A or S163E mutants CDH1. d IB analysis of indicated proteins derived from MCF-7 cells expressing either WT CDH1, S163A or S163E mutants CDH1 and treated with 1 μM palbociclib for 3 days. e IB analysis of GST pull-down precipitates derived from 293T cells transfected with GST-PIN1 and HA-CDH1 mutants for 36 h. f NMR analysis of phosphorylated peptide bound to PIN1. Average chemical shift perturbation in PIN1 backbone amide resonances on the binding of the CDH1 phosphopeptide. g Overlay of two-dimensional (2D) 1 H- 15 N Heteronuclear single quantum coherence (HSQC) spectrum from the backbone of R17, S18, W34, and E35, and the W34 sidechain of 15 N-labeled PIN1 (blue) and its complex with the CDH1 phosphopeptide (orange). h HADDOCK model demonstrating putative interaction between the CDH1 phosphopeptide shown as red sticks and PIN1 WW (magenta) and PPIase domain (cyan; PDB: 1PIN). i Overlay of 13 C-HSQC spectra acquired on free peptide (red) and its complex with PIN1 (green). The peak volumes were used to derive isomer population estimates. j DIA-MS analysis of the relative abundance of the peptide containing phosphorylation site of CDH1-S163 derived from WT or PIN1 KO MCF-7 cells stably expressing HA-CDH1. k IB analysis of indicated immunoprecipitates derived from WT or PIN1 KO MCF-7 cells expressing either WT CDH1, S163A or S163E mutants CDH1 and pulled down by anti-HA antibody. Input is 5% of the total lysates used in IP. l Schematic diagram illustrating PIN1-catalyzed trans to cis prolyl-isomerization of the CDH1-pS163-P motif. The images were representative images from 3 independent experiments ( a – e , k ). Source data are provided as a file.

Article Snippet: The purified HA-CDH1 proteins were then incubated with 500 uM of ATPγS (Abcam, ab138911) and 0.5 ug of recombinant human cyclin D1 + CDK4 proteins (Abcam, ab55695) in the kinase reaction buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35, pH 7.5) for 30 min at room temperature.

Techniques: Transfection, Construct, In Vitro, Kinase Assay, Expressing, Derivative Assay, Binding Assay, Labeling, Stable Transfection

Journal: Nature Communications

Article Title: Reciprocal antagonism of PIN1-APC/C CDH1 governs mitotic protein stability and cell cycle entry

doi: 10.1038/s41467-024-47427-w

Figure Lengend Snippet: a IB analysis of immunoprecipitates from WT and PIN1 -KO MDA-MB-231 cells treated with Palbociclib. b Dot plots of FACS for PIN1 -KO or WT MCF-7 cells stably expressing the APC/C-degron reporter and treated with AApin (1.5 μM ATO + 15 μM ATRA) for 3 days. Dots color, reporter levels. c Tracking cell division and cell death of MCF-7 cells in response to Palbociclib (4 μM), Sulfopin (10 μM) or AApin (1.5 μM ATO + 15 μM ATRA). d Frequency of G0/G1 arrest in MCF-7 cells from ( c ). n > 90 cells. The error bar indicates 95% confidence interval determined by bootstrapping. Detection of endogenous PIN1-CDH1 ( e ) and CDK4-CDH1 interactions ( f ) by PLA in the indicated cells treated with combination of 1 μM Palbociclib and 10 μM Sulfopin for 3 days. Nucleus were stained by DAPI. Scale bars, 5 μm. (Right) Quantification of PLA signals. n > 30 cells. Data are analyzed by unpaired two-sided t -test. p values are shown ( d – f ). g IB analysis for indicated proteins from WT and CDH1 -KO MCF-7 cells treated with combination of 1 μM Palbociclib and AApin (ATO (0.5, 1, 1.5, 2 μM) + ATRA (5, 10, 15, 20 μM)) for 3 days. h IB analysis for indicated proteins derived WT and CDH1 -KO BT-549 cells treated with 5 μM Sulfopin, 2.5 μM Palbociclib or their combination for 3 days. i CHX assay for indicated proteins from WT and CDH1 -KO MDA-MB-231 cells pre-treated with combination of 10 μM Sulfopin and 1 μM Palbociclib for 36 h followed by 50 µg/ml CHX for the indicated time. j IB analysis of ubiquitinated PIN1 from WT and CDH1 -KO MDA-MB-231 cells treated with 1 μM Palbociclib and 10 μM Sulfopin for 3 days and 2 μM MG132 for last 12 h and pulled down by Ni-NTA agarose. k , l Schematic diagrams showing the reciprocal inhibition of PIN1-APC/C CDH1 governs cell-cycle entry and exit. The images were representative images from 3 independent experiments ( a , b , e – j ). Source data are provided as a file.

Article Snippet: The purified HA-CDH1 proteins were then incubated with 500 uM of ATPγS (Abcam, ab138911) and 0.5 ug of recombinant human cyclin D1 + CDK4 proteins (Abcam, ab55695) in the kinase reaction buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35, pH 7.5) for 30 min at room temperature.

Techniques: Stable Transfection, Expressing, Staining, Derivative Assay, Inhibition

Journal: Lung India : Official Organ of Indian Chest Society

Article Title: Evaluation of Cyclin D1 protein and its association with the clinicopathological characteristics and prognosis of lung cancer: A retrospective study from Southern Kerala, India

doi: 10.4103/lungindia.lungindia_257_24

Figure Lengend Snippet: Clinical and pathological characteristics

Article Snippet: Monoclonal antibody against human cyclin D1 protein (Cat# sc-20044) was purchased from Santa cruz (USA).

Techniques: Expressing

Journal: Lung India : Official Organ of Indian Chest Society

Article Title: Evaluation of Cyclin D1 protein and its association with the clinicopathological characteristics and prognosis of lung cancer: A retrospective study from Southern Kerala, India

doi: 10.4103/lungindia.lungindia_257_24

Figure Lengend Snippet: Immunohistochemistry images of cyclin D1 expression in lung cancer tissues and normal tissues. (a). LUAD, (b). LUSC, (c). NSCLC, (d). PDC, (e). and NMC, (f). Graphical representation of individual cyclin D1 H scores for each histology type

Article Snippet: Monoclonal antibody against human cyclin D1 protein (Cat# sc-20044) was purchased from Santa cruz (USA).

Techniques: Immunohistochemistry, Expressing

Journal: Lung India : Official Organ of Indian Chest Society

Article Title: Evaluation of Cyclin D1 protein and its association with the clinicopathological characteristics and prognosis of lung cancer: A retrospective study from Southern Kerala, India

doi: 10.4103/lungindia.lungindia_257_24

Figure Lengend Snippet: Clinicopathological characteristics and cyclin D1 expression

Article Snippet: Monoclonal antibody against human cyclin D1 protein (Cat# sc-20044) was purchased from Santa cruz (USA).

Techniques:

Journal: Lung India : Official Organ of Indian Chest Society

Article Title: Evaluation of Cyclin D1 protein and its association with the clinicopathological characteristics and prognosis of lung cancer: A retrospective study from Southern Kerala, India

doi: 10.4103/lungindia.lungindia_257_24

Figure Lengend Snippet: Relationship between the expression of cyclin D1 and the prognosis of lung cancer

Article Snippet: Monoclonal antibody against human cyclin D1 protein (Cat# sc-20044) was purchased from Santa cruz (USA).

Techniques: Expressing

Journal: Lung India : Official Organ of Indian Chest Society

Article Title: Evaluation of Cyclin D1 protein and its association with the clinicopathological characteristics and prognosis of lung cancer: A retrospective study from Southern Kerala, India

doi: 10.4103/lungindia.lungindia_257_24

Figure Lengend Snippet: Kaplan–Meier survival curves at 24 months based on cyclin D1 expression status

Article Snippet: Monoclonal antibody against human cyclin D1 protein (Cat# sc-20044) was purchased from Santa cruz (USA).

Techniques: Expressing