Journal: Endocrinology

Article Title: Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity.

doi: 10.1210/endo.140.11.7145

Figure Lengend Snippet: FIG. 2. T3 stimulates cyclin E-associated kinase activity. Cell ex- tracts were prepared at the indicated times after culturing of GC cells in the presence or absence of T3. Cyclin E immunoprecipitates were assayed for kinase activity in the presence of histone H1 and [g-32P]ATP as described in Materials and Methods. Kinase reactions were subjected to SDS-PAGE followed by autoradiography. Results shown are representative of two independent experiments. A, Auto- radiograms of cyclin E-cdk phosphorylation of histone H1. B, Quan- titative analysis of the results shown in (A). The results were quan- titated with a PhosphorImager and values expressed as cyclin E-associated kinase activity of cells cultured in the presence of T3 relative to that of cells cultured in the absence of T3. Kinase activity obtained by performing the experiment in the absence of anti-cyclin E antibodies was substracted from its corresponding experimental group. Each time point represents the kinase activity from four in- dividual dishes. Data are expressed as mean 6 SD n 5 8. Statistical significance between the different groups was shown by ANOVA followed by Fisher’s PLSD (P , 0.05).

Article Snippet: Immunoprecipitation, in vitro kinase assay, and Western blot analysis For immunoprecipitation, either 400 mg (H1 phosphorylation) or 100 mg (GST-Rb phosphorylation) of protein extract were incubated with 20 ml of Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 C. The proteins binding nonspecifically to protein A/G Plus-Agarose were pelleted by centrifugation at 4,000 3 g for 5 min and the supernatants incubated for 3 h on ice with 4 mg of anti-cyclin E (sc-481) or anti-cyclin D1 (sc-450) for histone H1 or GST-Rb phosphorylation, respectively.

Techniques: Activity Assay, SDS Page, Autoradiography, Phospho-proteomics, Cell Culture

Journal: Nature Communications

Article Title: Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity

doi: 10.1038/ncomms12632

Figure Lengend Snippet: ( a ) Schematic diagram of GSK3β phosphorylation and β-TrCP-binding motifs and various mutants of PD-L1 expression constructs. PD-L1 was separated into ECD and ICD. SP, signal peptide; TM, transmembrane domain. The numbers indicate amino-acid positions. ( b ) In vitro GST pull-down assay of non-glycosylated PD-L1 and GSK3β. ( c ) Co-immunoprecipitation (co-IP) measuring the interaction of GSK3β and PD-L1 4NQ. Schematic diagram of PD-L1 4NQ deletion or truncation mutants showing on the left. Positions of glycosylation sites were labelled with red colour. The numbers indicate amino-acid positions. ( d ) Immunocomplex kinase assay measuring PD-L1 phosphorylation by GSK3β. Coomassie blue staining showing equal loading amount of GST-PD-L1. CA, constitutive activation mutant (S9A); KD, kinase dead (K85A) mutant; WT, wild type. ( e ) Western blot analysis of phosphorylation of PD-L1 protein at T180 and S184 sites by phospho-T180 and -S184 PD-L1 antibodies, respectively. EV, empty vector. ( f ) Time-lapse microscopy image (at 12 h) showing the dynamic interaction between PD-L1 and PD-1 at the last time point. The kinetic graph showed the quantitative binding of green fluorescent labelled PD-1/Fc protein on PD-L1 WT, 3SA or 4NQ expressing BT549 cells at every hour time point (right). Scale bar, 100 μm. ( g ) T-cell-meditated tumour cell-killing assay in PD-L1 WT or 3SA-expressing BT549 cells. Representative phase, red fluorescent (nuclear-restricted RFP), and/or green fluorescent (Caspase 3/7 substrate)-merged images of PD-L1 WT- or PD-L1 3SA-expressing cells and activated T-cell co-cultures at 96 h. Green fluorescent cell was counted as dead cell. The quantitative ratio of dead cells showed in bar graph (right). Scale bar, 100 μm. ( h ) The tumour growth of mouse PD-L1 WT- or PD-L1 3SA-expressing 4T1 cells in BALB/c mice. Quantification of tumour volume is shown on the right and representative images of tumours are shown on the left. n =7 mice per group. Con, vector control; WT, PD-L1 WT; 3SA, PD-L1 3SA. * P <0.05 is statistically significant as shown by Student's t -test. All error bars are expressed as mean±s.d. of three independent experiments.

Article Snippet: Briefly, antibodies against T180 and S184 phosphorylation sites of PD-L1 were generated in LifeTein (Somerset, NJ, USA).

Techniques: Phospho-proteomics, Binding Assay, Expressing, Construct, In Vitro, Pull Down Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Glycoproteomics, Kinase Assay, Staining, Activation Assay, Mutagenesis, Western Blot, Plasmid Preparation, Time-lapse Microscopy, Control