Anti flag rabbit mab

Cells cultured in cell coverslip in a 24-well plate were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized by 0.5% Triton X-100, and then blocked with 5% BSA. The treated cells were incubated with primary anti-FLAG Rabbit mAb (#14793, Cell Signaling Technology, Boston, MA, USA) overnight, and then incubated with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, DyLight 488 (#35553, Thermo Fisher, Sunnyvale, CA, USA) for 1 h. Finally, the coverslips were counterstained with DAPI, loaded on slide, sealed by nail polish, and visualized under a confocal laser-scanning microscope (Leica TCS SP8, Leica, Weztlar, Germany). Red fluorescence protein dsRed expression in cells was directly detected by microscope.

For western blotting, the input and IP samples were resolved by 7% homemade
SDS–PAGE protein gels [50 (link)], transferred
onto nitrocellulose membrane, blocked with 10% skim milk in 1× Tris-buffered
saline, 0.1% Tween 20 (1× TBST), probed overnight at 4°C with appropriate
primary (mouse anti-cMyc 9E10-horseradish peroxidase, mouse anti-FLAG M2, mouse
anti-HA; all Sigma–Aldrich or anti-FLAG rabbit mAb; Cell signalling) and then
for 2 h at room temperature with secondary antibodies (polyclonal goat
anti-mouse IgG-HRP or polyclonal goat anti-rabbit IgG-HRP; both Agilent Dako) in
2% skim milk in 1× TBST. The membranes were washed with 1× TBST and the specific
proteins were visualized using Clarity Western ECL substrate (Bio-Rad) on
ChemiDoc XRS+ System (Bio-Rad).