FIGURE 1:

Modification of lamin A by SUMO1 or SUMO2 in vitro and in Cos-7 cells. (A) Exogenous mature myc-lamin A is modified by SUMO1 and SUMO2. Cos-7 cells were transfected to express the indicated constructs for 36 h, then lysed, incubated with Ni²⁺ beads to recover His-SUMO and lamin A (which has a natural His tag), resolved by SDS–PAGE, and immunoblotted with antibodies to myc (α-myc); n = 3. (B) Recombinant purified mature (mA), precursor (pA), or Δ50 (Δ50) lamin A tail-domain polypeptides were incubated with SUMO1, E1, and E2 with or without ATP for 3 h at 30°C. Reactions were resolved by SDS–PAGE in duplicate and either stained with Coomassie blue or immunoblotted first with antibodies to the T7 tag (α-T7) and then stripped and reprobed with antibodies to SUMO1 (α-SUMO1); n = 3. Unmodified lamin A tails are marked by an asterisk and SUMO1-modified lamin A tails by a double asterisk. (C) Mature lamin A tails were incubated with either SUMO1 or SUMO2 (or both) along with E1, E2, and ATP for 1 h at 30°C and then immunoblotted with antibodies specific for SUMO1 (α-SUMO1), SUMO2 (α-SUMO2), or lamin A tail (α-Lamin A); n = 3.