Fig. 3.

Identification of the actual TM domain by in situ proteinase K digestion assay. Untransfected Cos-7 cells (A) and Cos-7 cells transfected with pEGFP (B) or expression vectors coding for selected Myc and EGFP double-tagged SUN4 proteins (C,D) were subjected to proteinase K digestion under different membrane permeabilization conditions and processed for immunoblotting (lanes 2–4; the first lanes show controls with untreated cells). (A) Analysis of PDI and lamin A/C in untransfected cells and (B) of EGFP in pEGFP-transfected cells. (C) Cells expressing Myc and EGFP double-tagged full-length SUN4 or C-terminally truncated SUN4 were tested for proteinase digestion behavior using anti-Myc and anti-GFP antibodies. (D) Analysis of SUN4 versions with deletions of either HM1, HM2 or both. Images are representative of at least two experimental repeats. KHM, KHM buffer.