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: Frontiers in Neuroscience

Article Title: LRRK2 Phosphorylation, More Than an Epiphenomenon

doi: 10.3389/fnins.2020.00527

Figure Lengend Snippet: Reported LRRK2 phosphorylation sites.

Article Snippet: 979 (Serine) , 2 (Gloeckner et al., ; Muda et al., ) , In vitro autokinase assay followed by mass spectrometric analysis Phosphopeptide enrichment by PKA followed by mass spectrometric analysis , Constitutive (Gloeckner et al., ) PKA phosphorylation (Muda et al., ) , No , No effect described.

Techniques: Phospho-proteomics, In Vitro, Purification, Cell Culture, Mass Spectrometry, Affinity Purification, Mutagenesis, Activity Assay, Binding Assay, Recombinant, Transfection

Journal: Frontiers in Neuroscience

Article Title: LRRK2 Phosphorylation, More Than an Epiphenomenon

doi: 10.3389/fnins.2020.00527

Figure Lengend Snippet: Phosphoregulation of LRRK2. Phosphoregulation of LRRK2 protein put together a lot of different partners, and some of those partners can also be regulated by LRRK2 itself. On the upstream regulation, the inhibitory phosphatases are localized on the left and the activating kinases are localized on the right. Kinases and phosphatases are implicated in the regulation of the N-ter phosphorylation sites (S910/935/955/973). N-ter sites and S1444 are phosphorylated by PKA while LRRK2 is also able to regulate the activity of PKA by a direct interaction with its ROC domain or by an indirect manner, by acting on the phosphodiesterase 4 (PDE4). PPP1CA has been confirmed to act on LRRK2. The holoenzyme PP2A could regulate the phosphorylation at S1292. The phosphorylation of the N-ter sites allows the interaction with 14-3-3. If phosphorylated by PAK6, the binding to LRRK2 is abolished. RAB29 interacts with LRRK2 in the Trans-Golgi network; this interaction leads to an increased phosphorylation of the N-ter and the kinase activity of LRRK2. LRRK2 can phosphorylate RAB29 and avoid LRRK2 activation, creating an inactivation loop (Nichols et al., ; Li et al., ; Dzamko et al., ; Lobbestael et al., ; Chia et al., ; Steger et al., ; Purlyte et al., ).

Article Snippet: 979 (Serine) , 2 (Gloeckner et al., ; Muda et al., ) , In vitro autokinase assay followed by mass spectrometric analysis Phosphopeptide enrichment by PKA followed by mass spectrometric analysis , Constitutive (Gloeckner et al., ) PKA phosphorylation (Muda et al., ) , No , No effect described.

Techniques: Phospho-proteomics, Activity Assay, Binding Assay, Activation Assay