Fig. 4.
Emerin intermolecular association in cells – deletions reveal positive and negative elements. (A) Schematic of GFP–emerin polypeptides studied in vivo. LEM, LEM domain; APC-L, APC-like homology domain; TM, predicted transmembrane domain. (B) GFP–emerin and Flag–emerin associate in HeLa cells. Sonicated (total) whole cell lysates (WCLT) from HeLa cells 24 h post-co-transfection with GFP–emerin (or GFP) and Flag–emerin were immunoprecipitated (IP) using GFP antibodies, resolved by SDS-PAGE (5% or 10%) alongside input lysates (1%), probed with Flag antibodies, then stripped and reprobed for GFP. Wild-type (WT) GFP–emerin specifically co-precipitated wild-type Flag–emerin (n = 2). Association was undetectable when both constructs lacked AR (ΔAR/ΔAR; n = 1) or R (ΔR/ΔR; n = 2). (C–F) GFP–emerin and Flag–emerin associate in HEK293T cells. (C,D) Sonicated ‘total’ whole cell lysates (WCLT) from HEK293T cells 24 h after co-transfection with wild-type Flag–emerin plus GFP or GFP–emerin (WT, ΔL, ΔA, ΔR or ΔAR) were immunoprecipitated using anti-GFP antibodies (IP: GFP). Precipitates (2–2.5%) and input control lysates (1%) from cells co-expressing both WT constructs were resolved by SDS-PAGE, immunoblotted for Flag (WB: Flag), stripped and reprobed for GFP (WB: GFP). Results (C) were quantified by densitometry and presented as a graph (D) as the amount of wild-type Flag–emerin precipitated per GFP–emerin mutant, relative to wild-type GFP–emerin. *P<0.05, **P<0.005 (n = 3). Results are mean±s.e.m. (E,F) Residues 67–108 (ΔM) are not essential for emerin–emerin association in HEK293T cells. Equal concentrations of whole cell lysates from HEK293T cells 24 h post co-transfection with GFP–emerin and Flag–emerin (both wild-type or both ΔM) were immunoprecipitated with anti-GFP antibodies, resolved by SDS-PAGE (2.5%) with input lysate controls (1%), immunoblotted for Flag or GFP (E), and quantified (F) as the Flag-to-GFP ratio for ΔM/ΔM, relative to WT/WT. Results are mean±s.e.m. (n = 2). (G,H) Immunoprecipitation from HEK293T cells that co-expressed two deletion constructs. Precipitates (2.5%; IP: GFP) and corresponding input lysates (1%; Inputs) were resolved separately, immunoblotted for Flag (WB: Flag), stripped and reprobed for GFP (WB: GFP). Black lines separate sections from same blot. Results (G) were quantified (H) as the ratio of Flag–emerin for each GFP–emerin construct, relative to cells expressing WT/WT. Results are mean±s.e.m. ΔAR/ΔR and ΔAR/ΔA were each tested twice (n = 2); others three times (n = 3). *P<0.05; **P<0.005.