Rabbit anti flag polyclonal affinity antibody

HEK293T cells were co-transfected with pcDNA3-HA-Ikzf1 (WT or L132P mutant) and pFlagCMV2-Ikzf1 or pcDNA3.1+/C-DYK-IKZF3 (GenScript) using Effectene transfection reagent (Qiagen). Cell lysates were prepared 20h after transfection in lysis buffer (50mM Tris [pH 7.4], 150mM NaCl, 2mM EDTA, 0.5% Triton X-100, and halt protease and phosphatase inhibitor cocktail (Thermo fisher scientific). Total protein (500μg) was incubated with rabbit anti-FLAG polyclonal affinity antibody (Sigma). After 2h incubation at 4 °C on a rotating wheel, 50μl Protein A/G-agarose beads (Pierce) were added to each reaction and incubation was continued for another hour. Beads were washed three times with lysis buffer, samples were prepared and separated on a NuPAGE® Novex® 4-12% Bis-Tris Protein Gels (Life Technology). Subsequent western blot analysis was performed with the mouse anti-HA monoclonal antibody (Covance), and rabbit anti-FLAG polyclonal affinity antibody.

NIH3T3 cells (0.8-1×10⁵) were seeded onto cover slips in 6 well plates. The next day, cells were transfected with the indicated plasmid using Effectene (Qiagen) according to the manufacturer’s instructions. The cells were washed twice in PBS, fixed for 10 min in 4% paraformaldehyde and permeabilized for 15 min in 0.1% Triton X-100 in PBS at RT. The cells were then incubated for 30 min in blocking buffer (PBS with 10% FBS and 0.1% Triton X-100) then incubated for 2h with a mouse anti-HA monoclonal antibody (Covance), and/or a rabbit anti-Flag polyclonal affinity antibody (Sigma). The cells were washed with PBS and incubated for 1h with goat anti-mouse Alexa Fluor 488 (Life Technologies) and/or goat anti-rabbit Alexa Fluor 568 (Life Technologies) conjugated secondary antibodies in blocking buffer. Cells were washed again and stained with DAPI for 5 minutes before a final wash and were then mounted on slides using VECTASHIELD Mounting Medium (Vector Laboratories). Images were collected using ZOE fluorescent cell imager (Bio-Rad) original magnification 175x. Red and green channels were cropped and merged after acquisition using ImageJ software.