I tried and cloned the gene in the 3xFLAG-APEX2-NES vector. i am transfecting 1.5ug of each plasmid in HEK293T cells in 12 well plates at 80% confluency then after 48 hrs of transfection I prepared lysate using RIPA+PIC. on probing with flag antibody only empty vector control is expressed not the fused protein. I have confirmed the clone by sequencing also.

Please help me out with the problem of why fusion protein is not expressed.

thank you