Visualization of Hidden Epitopes at the Inactive X Chromosome
Immunodetection of nuclear antigens is often complicated by epitope masking, so that proteins known to function in the nucleus are sometimes not easily detected at their sites of action. Moreover, protein populations that are detected before unmasking can be very different to those seen after removal of nucleic acids. This is particularly true for components of the nuclear matrix, including those known to function at the inactive X chromosome. Here we describe an unmasking protocol that reveals previously undetected proteins at the inactive X chromosome in mouse fibroblasts.
Key wordsUnmasking nuclear proteins Nuclear matrix Immunofluorescence SAF-A CIZ1 Inactive X chromosome
We are grateful to Barbora Kriukelyte for the PTBP1 antibody staining and Justin Ainscough for his comments. This work was funded by ERS PhD scholarship as part of BBSRC White Rose Doctoral Training Partnership grant BB/M011151/1.
- 3.Ma N, Matsunaga S, Morimoto A, Sakashita G, Urano T, Uchiyama S, Fukui K (2011) The nuclear scaffold protein SAF-A is required for kinetochore-microtubule attachment and contributes to the targeting of Aurora-A to mitotic spindles. J Cell Sci 124(Pt 3):394–404. https://doi.org/10.1242/jcs.063347CrossRefPubMedPubMedCentralGoogle Scholar
- 4.Ridings-Figueroa R, Stewart ER, Nesterova TB, Coker H, Pintacuda G, Godwin J, Wilson R, Haslam A, Lilley F, Ruigrok R, Bageghni SA, Albadrani G, Mansfield W, Roulson JA, Brockdorff N, Ainscough JFX, Coverley D (2017) The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory. Genes Dev. https://doi.org/10.1101/gad.295907.117CrossRefGoogle Scholar
- 6.Smeets D, Markaki Y, Schmid VJ, Kraus F, Tattermusch A, Cerase A, Sterr M, Fiedler S, Demmerle J, Popken J, Leonhardt H, Brockdorff N, Cremer T, Schermelleh L, Cremer M (2014) Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci. Epigenetics Chromatin 7:8. https://doi.org/10.1186/1756-8935-7-8CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Chen CK, Blanco M, Jackson C, Aznauryan E, Ollikainen N, Surka C, Chow A, Cerase A, McDonel P, Guttman M (2016) Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science 354(6311):468–472. https://doi.org/10.1126/science.aae0047CrossRefPubMedPubMedCentralGoogle Scholar