Purification of proteins containing the C-terminal domains of A-type and B-type lamins fused to GST.
(A) Schematic representation of human lamin A secondary structure. Downstream of the rod domain (dark gray boxes), the C-terminal end contains the NLS (black boxes) and an Ig-like domain (Ig fold, streaked boxes). Peptides of the C-terminal tails of A-type and B-type lamins used here, lamin A (LA, aa 389–646), prelamin A (preLA, aa 389–664), progerin (Pgn, aa 389-Δ-664), lamin C (LC, aa 389–572) and lamin B1 (LB1, aa 391–586) are indicated. Positions of specific C-terminus of lamin C (aa 566–572) and the 50 amino acids deletion in the progerin mutant (Δ607–656) are indicated.
(B) Fusion proteins. GST-C-ter lamin B1 (B1), lamin A (A), lamin C (C), prelamin A (pA), and progerin (Pgn) were resolved by SDS-PAGE and stained with Coomassie blue. (C) Immunoblotting analysis of the GST-C-ter prelamin A (pA). Samples containing 100 ng of GST-C-ter pA (lanes 7 and 8) were analyzed by 12.5% SDS-PAGE followed by immunoblotting using either rabbit polyclonal antibodies directed against the NLS of lamin A/C (αNLS, lane 8) or goat antibodies raised against the last amino acids of WT prelamin A (αC-ter pA, lane 7).