Analysis of Ser/Thr Kinase HASPIN-Interacting Proteins in the Spermatids
Abstract
:1. Introduction
2. Results
2.1. Screening of Proteins That Interact with HASPIN in a Mouse Testis cDNA Library Using Two-Hybrid System
2.2. Northern Blotting of Genes Interacting with HASPIN
2.3. Binding Evaluation of HASPIN and Proteins Screened by Two-Hybrid System in Cultured Cells
2.4. Expression of KPNA6/Importin α6 in the Testis
2.5. Expression of CENPJ/CPAP in the Testis
2.6. Expression of C1QBP/HABP1 in the Testis
2.7. Phosphorylation Target Protein of HASPIN
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Yeast Two-Hybrid System
4.3. Northern Blots and DNA Sequencing
4.4. RT-PCR
4.5. Western Blotting
4.6. Antibody Preparation
4.7. Expression Vectors
4.8. Transfection
4.9. Immunostaining
4.10. Immunoprecipitation
4.11. In Vitro Kinase Assay
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Registrated Gene Name (NCBI Accetion Number) | Domain in Cloned Gene | Number Of Clones Obtained |
---|---|---|
CenpJ/CPAP (NP_001014996) | Glycine repeat | 5 |
Kpna6/importin alpha 6 (NP_032494.3) | Whole | 2 |
Morn2 (NP_001347369) | MORN motif | 2 |
C1qbp/HABP1 (NP_031599.2) | Whole | 1 |
Ranbp9/RanBPM (NP_001391577.1) | SPRY domain | 1 |
Tcp10/t-complex protein 10 (X58170) | Glycine repeat | 1 |
Aip (NP_001263213.1) | Tetratricopeptide repeat | 1 |
Lims1 (NP_001180232.1) | LIM domain | 1 |
Pdlim5 (NP_001177781.1) | LIM domain | 1 |
Mad2l2 (NP_001292349.1) | HORMA domain | 1 |
Name | Reference |
---|---|
anti-KPNA6-rabbit IgG | this study |
anti-CENPJ-rabbit IgG | this study |
anti-C1QBP rabbit IgG | Muta et al. [69] |
anti-HASPIN rabbit serum | Tanaka et al. [2] |
Integrin β1 antibody | SantaCruz Biotechnology (SC-8978) |
Anti-FLAG antibody | Sigma-Aldrich (F3165) |
monoclonal antibody TRA98 | Fukuda et al. [14,70] |
monoclonal antibody TRA54 | Pereira et al. [17] |
rabibit anti-ACTB antibody | Cell Signaling (4967) |
anti-rabbit IgG HRP-linked whole Ab (from donkey) | Amersham Biosciences (NA934V) |
rabbit anti-rat IgG HRP-secondly antibody | DAKO (P0450) |
donkey anti-rabbit IgG, biotinylated species specific antibody | Amersham Biosciences (SAB3700865) |
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Maeda, N.; Tsuchida, J.; Nishimune, Y.; Tanaka, H. Analysis of Ser/Thr Kinase HASPIN-Interacting Proteins in the Spermatids. Int. J. Mol. Sci. 2022, 23, 9060. https://doi.org/10.3390/ijms23169060
Maeda N, Tsuchida J, Nishimune Y, Tanaka H. Analysis of Ser/Thr Kinase HASPIN-Interacting Proteins in the Spermatids. International Journal of Molecular Sciences. 2022; 23(16):9060. https://doi.org/10.3390/ijms23169060
Chicago/Turabian StyleMaeda, Naoko, Junji Tsuchida, Yoshitake Nishimune, and Hiromitsu Tanaka. 2022. "Analysis of Ser/Thr Kinase HASPIN-Interacting Proteins in the Spermatids" International Journal of Molecular Sciences 23, no. 16: 9060. https://doi.org/10.3390/ijms23169060