The Neuronal-Specific SGK1.1 (SGK1_v2) Kinase as a Transcriptional Modulator of BAG4, Brox, and PPP1CB Genes Expression
Abstract
:1. Introduction
2. Results and Discussion
2.1. Screening the Subtracted cDNA Library Identified Six Independent and Differentially Expressed Clones
2.2. Anti-Brox Antibody (Brox-Ab) Specificity
2.3. Validation of Genes Identified by Subtraction Screening
Gene | SGK1.1S515D | SGK1.1K220A | p Value |
---|---|---|---|
BAG4 | 96.5 ± 62.1 | 41.3 ± 27.3 | 0.001 |
Brox | 178.8 ± 101.9 | 81.7 ± 61.6 | 0.001 |
LYCAT | 319.9 ± 210.1 | 196.6 ± 117.4 | 0.029 |
PPP1CB | 2492.9 ± 1625.4 | 1528.0 ± 832.2 | 0.023 |
RDX | 1355.0 ± 961.6 | 829.7 ± 638.5 | 0.042 |
C6ORF62 | 4684.2 ± 2856.7 | 4341.8 ± 3026.0 | 0.715 |
2.4. Immunolocalization in HeLa Cells
3. Experimental Section
3.1. Site Directed Mutagenesis
3.2. Cell Culture and Transfection
3.3. mRNA Extraction from Cells, Double Stranded cDNA Synthesis and PCR Amplification
3.4. ds-cDNA Subtraction and Library Construction
3.5. Dot-Blot Screening
3.6. DNA Sequencing and Sequence Identification
3.7. Gene Expression Analysis by Quantitative RT-PCR (qRT-PCR)
3.8. Plasmid Construction and Preparation of Recombinant Brox Protein
3.9. Production and Purification of Polyclonal Antibodies against the Brox
3.10. Protein Expression Analysis by Western Blot
3.11. Immunofluorescence Microscopic Analyses
3.12. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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González-Fernández, R.; Ávila, J.; Arteaga, M.F.; Canessa, C.M.; Martín-Vasallo, P. The Neuronal-Specific SGK1.1 (SGK1_v2) Kinase as a Transcriptional Modulator of BAG4, Brox, and PPP1CB Genes Expression. Int. J. Mol. Sci. 2015, 16, 7462-7477. https://doi.org/10.3390/ijms16047462
González-Fernández R, Ávila J, Arteaga MF, Canessa CM, Martín-Vasallo P. The Neuronal-Specific SGK1.1 (SGK1_v2) Kinase as a Transcriptional Modulator of BAG4, Brox, and PPP1CB Genes Expression. International Journal of Molecular Sciences. 2015; 16(4):7462-7477. https://doi.org/10.3390/ijms16047462
Chicago/Turabian StyleGonzález-Fernández, Rebeca, Julio Ávila, María F. Arteaga, Cecilia M. Canessa, and Pablo Martín-Vasallo. 2015. "The Neuronal-Specific SGK1.1 (SGK1_v2) Kinase as a Transcriptional Modulator of BAG4, Brox, and PPP1CB Genes Expression" International Journal of Molecular Sciences 16, no. 4: 7462-7477. https://doi.org/10.3390/ijms16047462