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Plasmid construction. The fragment covering myelin basic protein 87-99 full-length coding sequence for Apr3 and its truncation without the predicted transmembrane and intracellular domain were amplified from HL-60 cDNA using the same forward primer and two different reverse primers listed as follows: 5′-TGGAATTCATGAAGACCAGCGCAGAGC-3′ (forward), 5′-GTCTCGAGAAAGTCTTGGCTTTTCGGCGC-3′ (reverse), 5′-CTCTCGAGAAGCCCTGGCGCATACAC-3′ (reverse). The PCR products and our previously constructed pcDNA3.1(+)-3×flag vector were digested with EcoRI and XhoI before ligation. The new constructions, designated as pcDNA3.1-3×flag-Apr3 and pcDNA3.1-3×flag-Apr3Δ, in which Apr3 or Apr3Δ are in-frame fused with 3×flag at N-terminus, are confirmed by sequencing.
Subcellular localization of Apr3. MCF-7 cells were plated onto glass coverslips and transfected with pcDNA3.1-3×flag-Apr3 and pcDNA3.1-3×flag-Apr3Δ using lipofectamine? 2000 (Invitrogen). Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde for 20 min and permeabilize with 0.2% Triton X-100 in PBS for 10 min. Blocking solution (1% BSA in PBS) was applied for a further 60 min. Then rabbit anti-flag (1:1000, Sigma) was applied overnight at 4 °C. Flag-tagged proteins and DAPI-stained nuclei were visualized by Fluorescence microscope (Olympus, Japan).