rabbit anti-mouse per2  (Alpha Diagnostics)

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 3. Phosphorylation of the human PER2 FASP region inhibits CK1 activity (A–D) NMR kinase assay for the WT FASP or indicated mutant peptides monitoring the reaction kinetics of priming phosphorylation at S662 by NMR. (E) Schematic of FASP alanine mutations and resulting discrete phosphostates. (F) Plot of priming rate constant (kprime) as a function of the possible successive phosphosites in the FASP. Error bars represent SEM from fits in (A)–(D). (G) ADP-Glo kinase assay with titration of FASP mutant peptides with mean and SD from 2 replicates, representative of n = 3 independent assays. Shaded area indicates 95% CI of the fit. (H) Data from (F) normalized by Vmax values calculated from the preferred kinetic model (see Figure S2; Table 1). (I) ADP-Glo kinase assay of hPER2 PAS-Degron peptide (see Figure S2B) with titration of pFASP peptides corresponding to 2 (2p) or 3 (3p) phosphoserines (see Figure S3A) with mean and SD from 2 replicates, representative of n = 3 independent assays. Shaded area indicates 95% CI of the fit. (J and K) Western blot and quantification of the phosphorylation of the FASP priming site (pS659 in mouse PER2). Full-length mouse PER2 was immunopre- cipitated from transfected HEK293T cells, dephosphorylated, and subjected to an in vitro kinase assay with 200 ng CK1 in the presence and absence of un- phosphorylated mouse PER2 FASP or human PER2 4pFASP peptides as indicated.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Phospho-proteomics, Activity Assay, Kinase Assay, Mutagenesis, Titration, Western Blot, Transfection, In Vitro

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 4. The human PER2 pFASP binds to the active site of CK1 (A) Surface representation of CK1 catalytic domain bound to ADP (PDB 5X17). Spheres, sulfate anions from the crystallization condition bound at anion binding sites as indicated. (B) Surface representation of the CK1 catalytic domain bound to a human PER2 3pFASP peptide (see Figure S3A). (C–E) Zoom of 3pFASP interactions within anion binding site 1 (C), the active site (D), and anion binding site 2 (E). (F) Structural alignment showing the main chain for the activation loop of CK1 WT (gray) and the tau (R178C) mutant (red) showing a clash of the 3pFASP (teal) with the conformation of the activation loop stabilized by the tau mutation. (G) Characterization of key interactions from molecular dynamics simulations that stabilize the 5pFASP product in the active site, site 1, site 2, and an additional anion binding site located proximal to site 2 (site 30). Histograms were computed based on distances sampled during the GaMD simulations.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Crystallization Assay, Binding Assay, Activation Assay, Mutagenesis

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 5. Kinase anchoring interactions with the PER2 CK1BD increase FASP phosphorylation and feedback inhibition of CK1 (A) Alignment of human and mouse PER1/2 proteins showing conservation of the disordered FASP region flanked by two highly conserved binding motifs (CK1BD-A/B) characterized as PONDR (predictor of natural disordered regions) minima and predicted to be a-helices by AlphaFold2. The priming site (S662, human PER2 numbering) is shown, with sequential phosphorylation sites indicated by asterisks.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Phospho-proteomics, Inhibition, Binding Assay

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 6. Circadian rhythms are shortened by small deletions in the conserved FASP domain of human PER2 (A) Schematic representation of select in-frame deletions within the human PER2 FASP region are separated into 2 classes: priming-disrupted (P2E17-50) or priming intact (P2E17-25). (B) Immunoblot of select priming-disrupted PER2::LUC mutants compared with WT PER2::LUC. Blot representative of two independent experiments. (C and D) Representative immunoblots for (C) priming-disrupted or (D) priming intact PER2 mutants. *nonspecific band. The Per2 allele in clone 17-3 has a frame- shifting mutation leading to the deletion of the untagged PER2. (E and F) Real-time bioluminescence traces of circadian rhythms from WT and mutant Per2 clones (E, priming-disrupted; F, priming intact) with quantification of mean period and SD from n = 3 cultures. Periods from mutant clones were compared with WT with an unpaired t test: ***, p < 0.001. Per2 priming intact mutants exhibited rhythms that were shortened to a lesser degree than the priming-disrupted mutants in (E), p < 0.05. In both graphs, the first peaks are aligned to show differences in period clearly. (G) Western blot and quantification of degradation of WT PER2::LUC and clone 17–50 after cycloheximide (CHX) treatment at time 0. Blot representative of two independent assays (n = 2). (H) Western blot and densitometric quantification of phosphorylation of de novo PER2 in WT PER2::LUC and clone 17–50 after protein depletion by 10 h CHX treatment and washout at time 0. Blot representative of two independent assays (n = 2).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Western Blot, Mutagenesis, Clone Assay, Phospho-proteomics

Journal: Molecular cell

Article Title: PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

doi: 10.1016/j.molcel.2023.04.019

Figure Lengend Snippet: Figure 7. The phosphorylated PER-Short domain of Drosophila PER binds CK1 site 1 to inhibit kinase activity (A and B) Western blot of dPER fragment (aa1–100) cleaved from full-length dPER (A) or a fragment containing the dPER CK1BD but lacking the PAS dimerization domain (B) co-expressed with DBT. Samples collected at indicated time points after kinase induction, followed by protein extraction and TEV protease cleavage. (C) Densitometric quantification of the hypophosphorylated band (n = 4). WT and S596A compared with two-way ANOVA with Sidak multiple comparisons test: * p < 0.05. (D) ADP-Glo kinase assay of CK1 on the human PER2 PAS-Degron substrate in the presence of indicated peptides from dPER. Data are mean and SD from 2 replicates, representative of n = 3 independent assays. (E) Structure of human CK1 (gray) bound to the dPER-Short peptide with pS589 (light green). (F) Close-up view of dPER pS589 coordinated by CK1 residues in anion binding site 1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-FLAG Millipore Sigma cat. # F1804 anti-mouse IgG HRP Millipore Sigma cat. #12-349 anti-PER1 Lee et al.4 GP62 anti-PER2 Lee et al.4 GP49 anti-guinea pig IgG HRP Thermo Fisher Scientific cat. # A18769 anti-Myc agarose conjugate Santa Cruz Biotechnology cat. # sc-40 AC a-Myc HRP Santa Cruz Biotechnology cat. # sc-40 HRP a-OctA HRP Santa Cruz Biotechnology cat. # sc-166355 HRP anti-Myc Santa Cruz Biotechnology cat. # sc-40 Rabbit polyclonal anti-mouse PER2 phospho-Ser659 Narasimamurthy et al.18 N/A anti-rabbit IgG HRP Bio-Rad cat. # 1706515 anti-mouse IgG HRP Bio-Rad cat. # 1706516 Bacterial and virus strains Escherichia coli DH5a NEB cat. # C2987H Escherichia coli BL21 (DE3) Rosetta2 Fisher cat. # 69041 all-in-one CRISPR adenovirus Jin et al.73 N/A Chemicals, peptides, and recombinant proteins 2pFASP peptide Biopeptide Co.

Techniques: Activity Assay, Western Blot, Protein Extraction, Kinase Assay, Binding Assay