Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: CKIɛ and CKIδ bind to and phosphorylate mPer proteins specifically. (A) Luciferase (control), mPer1, mPer2, mPer3, mCry1, mCry2, and mBMAL1 were separately synthesized in vitro by using TNT T7 Coupled Reticulocyte Lysate System (right panel) and then mixed with in vitro-synthesized HA-tagged CKIɛ or CKIɛ(KR) and incubated. Each of these mixtures was subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were detected by a Bio-Rad phosphorimager (left panel). (B) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. Each of the cell extracts was incubated with GST, GST-fused CKIɛ, or GST-fused CKIɛ(KR) (expressed in E. coli, and purified) and glutathione-Sepharose 4B. The beads were then washed with the incubation buffer. After resolution by SDS-PAGE, proteins were analyzed by immunoblotting with anti-Myc antibody (A-14). (C) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells. The cell extracts were subjected to immunoprecipitation with anti-Myc antibody (9E10). The washed beads were mixed with a kinase reaction buffer supplemented with GST-fused CKIɛ or GST-fused CKIɛ(KR), as well as [γ-32P]ATP, and incubated for 30 min. After resolution by SDS-PAGE, substrate phosphorylation was detected by a Bio-Rad phosphorimager. (D) Myc-tagged mPer1, mPer2, mPer3, mCry1, mCry2, or mBMAL1 was expressed in COS7 cells, together with HA-tagged CKIɛ, CKIɛΔC, CKIδ, or CKIɛ(KR). The cell lysates were subjected to immunoprecipitation with anti-HA antibody (12CA5). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14). (E) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, together with or without CKIδ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10), and the washed beads were resuspended in phosphatase buffer and incubated with or without (mock) 40 U of purified lambda phosphatase for 30 min. After resolution by SDS-PAGE, the immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Luciferase, Synthesized, In Vitro, Incubation, Immunoprecipitation, SDS Page, Purification, Western Blot

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: CKI-induced nuclear translocation of mPer3 depends on CKI-mediated phosphorylation and NLS of mPer3. (A) Myc-tagged mPer3 was expressed in COS7 cells, together with or without HA-tagged CKIɛ, CKIδ or CKIɛ(KR). The cells were fixed and incubated with primary antibodies (anti-Myc antibody [9E10] and anti-HA polyclonal antibody) and then with the appropriate secondary antibodies. The cells were examined by using a Zeiss Axiophoto. (B, left panel) Myc-tagged mPer3, mut7, or mutNLS (see the right panel) (0.9 μg of DNA) was expressed in COS7 cells, together with or without HA-tagged CKIɛ, CKIδ, or CKIɛ(KR) (0.1 μg of DNA). After being stained, the cells were classified into three categories in terms of location of Myc-tagged mPer proteins as nucleus (N > C), cytoplasm (N < C), or both nucleus and cytoplasm (N = C). The classifications “N > C” and “N = C” are indicated by dark gray and light gray boxes, respectively. More than 150 cells were examined, and the percentages of N > C and N = C are shown. Experiments were performed twice with similar results. (B, right panel) A PCR-generated mutation was made in a presumable mPer3 NLS (mutNLS) and compared to the wild-type (WT) sequence. WT mPer3 or mPer3mutNLS was expressed in COS7 cells, together with or without CKIɛ. The cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). (C) The cells were subjected to transfection and kept in a medium containing 1% serum, after the serum shock (50% for 2 h). Myc-tagged mPer3 was expressed in COS7 cells, together with or without CKIɛ. The cells were then lysed (or fixed for staining) 30, 36, 42, and 48 h after the serum shock (time = 0 h). In the left panel, cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). The electrophoretic retardation of the mPer3 protein bands results from phosphorylation. In the right panel, cells were classified after being stained into three categories with regard to the location of Myc-tagged mPer3: N > C, N < C, or N = C. The N > C and N = C classifications are indicated by dark gray and light gray boxes, respectively. More than 250 cells were examined, and the percentages of the N > C and N = C groups are shown.

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Translocation Assay, Incubation, Staining, Generated, Mutagenesis, Sequencing, Western Blot, Transfection

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: In vivo association between mPer1 and CKIɛ. (A and B) Various mammalian cells were lysed in the incubation buffer. After the cell extracts were resolved by SDS-PAGE, they were analyzed by immunoblotting with anti-mPer1(N) antibody or anti-actin antibody (loading control) (A) or with anti-mPer1(C) antibody (B). (C) Myc-tagged mPer1, mPer2, or mPer3 was expressed in COS7 cells, and the cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10). These immunoprecipitates were analyzed by immunoblotting with anti-mPer1(C) antibody (upper panel) or anti-Myc antibody (A-14) (lower panel). (D) For coimmunoprecipitation experiments, NIH 3T3 cells were incubated in hypotonic lysis buffer and homogenized. Each of antibodies and protein G-Sepharose beads were added to the resulting supernatant. The beads were then washed with the hypotonic lysis buffer. The immunoprecipitates were analyzed by immunoblotting with anti-CKIɛ antibody. Anti-HA antibody and anti-Myc antibody were used as a nonrelevant antibody control.

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: In Vivo, Incubation, SDS Page, Western Blot, Immunoprecipitation, Lysis

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: Identification of potential CKIɛ phosphorylation sites on mPer3. (A) Amino acid sequences of the mammalian Per family. Amino acid identities and similarities are indicated by dark gray and light gray boxes, respectively. The conserved serine-threonine residues are indicated by open circles. (B) PCR-generated alanine mutations were made in the mPer3 conserved serine-threonine cluster and compared with the wild-type (WT) mPer3 sequence. (C and D) Wild-type (WT) mPer3 or each of various mutants of mPer3 (0.7 μg of DNA) was expressed in COS7 cells, together with or without CKIɛ (0.3 μg of DNA). The cell extracts were subjected to immunoblotting with anti-Myc antibody (A-14). Equal amounts of proteins were loaded. The electrophoretic retardation of the mPer3 protein bands results from phosphorylation. (E) Myc-tagged mut7 was expressed in COS7 cells, together with or without CKIɛ. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10) and incubated with or without (mock) purified lambda phosphatase. The immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody (A-14).

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Generated, Sequencing, Western Blot, Immunoprecipitation, Incubation, Purification

Journal:

Article Title: Control of Intracellular Dynamics of Mammalian Period Proteins by Casein Kinase I ? (CKI?) and CKI? in Cultured Cells

doi: 10.1128/MCB.22.6.1693-1703.2002

Figure Lengend Snippet: CKI promotes ubiquitination of mPer proteins. (A) To show ubiquitination of mPer proteins, each of Myc-tagged mPer proteins was coexpressed with HA-tagged ubiquitin in the presence or absence of CKIɛ, CKIδ, or CKIɛ(KR). The cell lysates were then subjected to immunoprecipitation with anti-Myc antibody (9E10). After resolution by SDS-PAGE, immunoprecipitates were analyzed by immunoblotting with anti-HA polyclonal antibody. The accumulation of high-molecular-mass species recognized by the anti-HA antibody indicates that Myc-tagged proteins become multiply ubiquitinated. (B) To study the metabolic stability of mPer proteins, each of Myc-tagged mPer proteins was expressed in COS7 cells, together with or without CKIɛ. The cells were incubated in methionine-cysteine-deficient medium and then pulsed with 35S-labeled methionine-cysteine. The cells were incubated for the indicated lengths of time in DMEM-10% fetal calf serum with or without MG-132. The cell lysates were subjected to immunoprecipitation with anti-Myc antibody (9E10). The immunoprecipitates were detected with a Bio-Rad phosphorimager.

Article Snippet: Anti-Myc monoclonal antibody (9E10) and rabbit anti-Myc polyclonal antibody (A-14) were purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-hemagglutinin (HA) polyclonal antibody was from Clontech.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Incubation, Labeling

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 1. Experiment setup. Blood: blood sampling, BP: blood pressure, Urine: urine sampling, Echo: echocardiography, CKD: chronic kidney disease, PBS: phosphate-buffered saline, P234: KISS1R antagonist peptide-234, LV: left ventricle, Op: operation. Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques: Sampling, Saline

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 2. The effects of the KISS1R antagonist peptide-234 on the development of CKD in 5/6 nephrectomized rats. (A) Serum urea concentration, (B) serum creatinine concentration, (C) creatinine clearance, (D) 24-h urinary protein excretion, (E) 24-h urinary creatinine excretion, and (F) 24-h urine volume at the endpoint. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham group (n = 7–8, One-Way ANOVA, Holm-Sidak post hoc test), $p < 0.05 vs. the week 5 values in the same group (n = 7–8, Two-Way Repeated Measures ANOVA, Holm-Sidak post hoc test). Creatinine clearance was calculated according to the standard formula (urine creatinine concentration [μM] × urine volume for 24 h [mL])/(serum creatinine concentration [μM] × 24 × 60 min). ¥At the endpoint, urine volume and creatinine concentration were measured at week 12 and serum creatinine concentration at week 13. Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques: Concentration Assay

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 3. The effects of the KISS1R antagonist peptide-234 on the echocardiographic parameters. (a) Representative M-mode images, (b) systolic posterior wall thickness (PWTs), (c) diastolic posterior wall thickness (PWTd), (d) Representative pulse wave and tissue Doppler images of mitral valve early flow velocity (e) and septal mitral annulus (e′) velocity, E) E/e′ ratio, (f) ejection fraction (EF). Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, #p < 0.05 vs. CKD (n = 7–9, One-Way ANOVA, Holm-Sidak post hoc test). $p < 0.05 vs. week 5 values in the same group (n = 7–8, Two-Way Repeated-Measures ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques:

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 4. The effects of the KISS1R antagonist peptide-234 on cardiomyocyte hypertrophy and interstitial fibrosis and molecular markers of heart failure at week 13. (a) Representative hematoxylin–eosin (HE)-stained slides at 100 × and 40 × magnifications and representative picrosirius red and fast green (PSFG)-stained slides at 20 × magnifcation (b) cardiomyocyte cross-sectional areas, (c) left ventricular collagen content, (d) A-type natriuretic peptide (Nppa), and (E) matrix metalloproteinase-9 (Mmp9) expressions in the left ventricles normalized to the ribosomal protein lateral stalk subunit P2 (Rplp2) gene expression. On the digital HE images, cardiomyocyte cross-sectional areas were measured in 100 selected cardiomyocytes on left ventricular sections cut on the same plane. The mean values of the collagen content of 10 representative PSFG-stained images were calculated and used for statistical evaluation in the case of each left ventricular slide. Scale bars represent 10 µm at the 100 × magnifed images, 20 µm at the 40 × magnifed images, and 50 µm at the 20 × magnifed images. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, #p < 0.05 vs. CKD (n = 7–8, one-way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques: Staining, Gene Expression

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 5. The effects of the KISS1R antagonist peptide-234 on the left ventricular expression of genes associated with inflammation and fibrosis. Relative gene expression of (a) interleukin-1 (Il1), (b), interleukin-6 (Il6), (c) tumor necrosis factor-α (Tnf), (d) connective tissue growth factor (Ctgf), (e) transforming growth factor-β (Tgfb), (f) collagen type 1 alpha 1 chain (Col1a1) and (g), collagen type 3 alpha 1 chain (Col3a1) normalized to the ribosomal protein lateral stalk subunit P2 (Rplp2) gene expression. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, #p < 0.05 vs. CKD (n = 7–8, One-Way ANOVA, Holm-Sidak post hoc test). Sham: sham- operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234.

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques: Expressing, Gene Expression

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 6. The effects of the KISS1R antagonist peptide-234 on the protein levels of KISS1R and ERK1/2 at week 13. Left ventricular protein levels and cropped representative Western blot imagines of (a) Kisspeptin receptor-1 (KISS1R, 40–140 kDa), (b) total ERK1 (44 kDa), (c) phospho-ERK1 (pERK1, 44 kDa), (d) pERK1/ERK1 ratio and (e) total ERK 2 (42 kDa), (f) phospho-ERK2 (pERK2, 42 kDa), (g) pERK2/ERK2 ratio. Values are presented as mean ± S.E.M., *p < 0.05 vs. sham (n = 7, One-Way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234. Images were captured with the Odyssey CLx machine and exported with Image Studio 5.2.5 software. The full-length Ponceau-stained membranes and the corresponding Western blot images are presented in the Supplementary Material (Figs. S3– S5).

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques: Western Blot, Software, Staining

Journal: Scientific reports

Article Title: The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model.

doi: 10.1038/s41598-023-41037-0

Figure Lengend Snippet: Figure 7. The effects of the KISS1R antagonist peptide-234 on apoptosis-associated gene expressions and protein levels in the left ventricles at week 13. Relative gene expression of (a) BCL2-associated X apoptosis regulator (Bax), (b) B-Cell CLL/lymphoma 2 apoptosis regulator (Bcl2), (c) Bax/Bcl2 ratio, and (d) caspase7 (Casp7) normalized to the ribosomal protein lateral stalk subunit P2 (Rplp2) gene expression. Left ventricular protein levels and cropped representative imagines of (e) BAX (20 kDa), (f) BCL2 (26 kDa), (g) BAX/BCL2 ratio, and (h) CASP 7 (35 kDa). Values are presented as mean ± S.E.M., *p < 0.05 vs. sham, #p < 0.05 vs. CKD vehicle group (n = 7–8 for RT-qPCR and n = 7 for Western blot measurements, One-Way ANOVA, Holm-Sidak post hoc test). Sham: sham-operated group, CKD: chronic kidney disease group, CKD + P234 D1: chronic kidney disease group treated with the lower dose (13 μg/day, dose 1) of KISS1R antagonist peptide-234, CKD + P234 D2: chronic kidney disease group treated with the higher dose (26 μg/day, dose 2) of KISS1R antagonist peptide-234. Images were captured with the Odyssey CLx machine and exported with Image Studio 5.2.5 software. The full-length Ponceau-stained membranes and the corresponding Western blot images are presented in the Supplementary Material (Figs. S6–S8).

Article Snippet: The time course and doses of P234 (Tocris Bioscience, Bristol, UK, catalog No.: 3881, molecular weight: 1295.42 g/mol; peptide sequence: Ac-DAla-Asn-Trp-Asn-Gly-Phe-Gly-D-Trp-Arg-Phe-NH2) were selected based on our preliminary data and previous studies26,44.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Software, Staining