Cy3 conjugated anti rabbit antibody

Cells were grown on poly-lysine-treated coverslips in 6-well plates. Each laminC-GFP amber mutant plasmid was cotransfected with the amber suppressor plasmid as transient transfection by the PEI-method. For the laminC-GFP amber mutant screen two samples were prepared with or without addition of 1 mM pBPA. 24 h post transfection (p.t.) the medium was replaced by fresh medium with or without pBPA. For the lmnC-T534-3xFlag cdk1 mutants, only one dish with medium with 1 mM pBPA was required. For the GFP-constructs, cells were washed two times with PBS, fixed for 15 min at RT with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS for 15 min at RT. Cells were incubated for 15 min with PBS containing 1 μg/ml DAPI. The different lmnC-T534-3xFlag constructs were immune-stained by fixing the cells with 4% paraformaldehyde, blocking with blocking buffer (3% BSA, 0.2% Triton X-100 in PBS), incubation with the primary antibody against the Flag-tag (1:1000 rabbit, F7425 Sigma-Aldrich) in blocking buffer for 2 h at RT, then washing 3 times for 5 min in PBS followed by incubation with a secondary Cy3-conjugated anti-rabbit antibody (1:1000 Jackson Immuno Research, 111-165-144) and 1 μg/ml DAPI for 1 h at RT. Thereafter, the coverslips were dried, mounted and analyzed by LSM (Zeiss LSM800/510, 40x or ×63 oil objective).