Haque, F., Lloyd, D.J., Smallwood, D.T., Dent, C.L., Shanahan, C.M., Fry, A.M., Trembath, R.C. and Shackleton, S.
Notes: To examine the putative role that SUN1, a nuclear envelope (NE) protein with a C-terminal SUN (Sad1/UNC-84 homology) domain, may play in the inner nuclear membrane, the murine SUN1 cDNA was subcloned into the pCI-neo Mammalian Expression Vector. Primers containing the myc or HA tag sequence were used to amplify the pCI-neo construct, adding tags both 5’ and 3’ of the SUN1 cDNA. Emerin, a marker for the NE lumen, was also subcloned into the pCI-neo Vector and a 3’ myc tag added. These constructs were translated and labeled with 35S in vitro using the TNT® T7 Quick Coupled Transcription/Translation System, and the radiolabeled proteins were used in a GST pull-down assay. In addition, NIH/3T3 cells were transiently transfected with myc-SUN1 constructs, fixed in methanol and then co-stained with anti-myc and anti-lamin A/C antibodies. (3511)