<p>To examine whether polymerized microtubules interact directly with Egr3 protein, we performed a microtubule-interaction assay. Tubulin from the bovine brain (5 μg/μl) was polymerized at 37°C and diluted to 0.2 μg/μl. Four microliters of in vitro translated lysates (NM or BC) and 50 μl of diluted MTs were incubated at room temperature for 30 min. The reaction was centrifuged at 100,000×g for 30 min to pellet the polymerized MTs. The supernatant (S) and pellet (P) were run on 10% SDS-PAGE and Western blotting was performed with anti-Egr3 or anti-Myc antibody which detects the epitope in the pcDNA3.1/myc-his vector. The antibodies were used at 1∶5000. The blots were stripped and tubulins were detected with anti-tubulin antibody. The data show that the presence of microtubules does not affect the quantity of Egr3 products in the supernatant and pellet, suggesting that there is no direct interaction between microtubules and Egr3.</p
