Tubulin-bound SEPT2 complexes inhibit binding of the microtubule-binding domain of MAP4 (C-MBD-MAP4) to tubulin. Equal amounts (2 μg) of purified tubulin were separated by 10% SDS-PAGE, transferred to nitrocellulose, and stained with Ponceau red. (A) Tubulin-containing strips were overlaid with increasing concentrations (0–4 μg/ml) of GST-tagged C-MBD-MAP4 directly (solid line; blot #1), or first overlaid with His-tagged SEPT2/6/7 (2 μg/ml) and then increasing concentrations of C-MBD-MAP4-GST (dotted line; blot #2). (B) Tubulin-containing strips were overlaid with increasing concentrations (0–4 μg/ml) of His-SEPT2/6/7 (1° overlay) and then with C-MBD-MAP4-GST (2 μg/ml; 2° overlay). (C) Tubulin strips were overlaid with increasing concentrations (0–4 μg/ml) of GST-tagged kinesin heavy chain (KHC-GST; 1° overlay) and then, with C-MBD-MAP4-GST (2 μg/ml; 2° overlay). (D) Tubulin was overlaid with an increasing concentration (0–6.4 μg/ml) of anti-polyGlu tubulin (mAb B3; 1° overlay) and then with C-MBD-MAP4-GST (2 μg/ml; 2° overlay). Tubulin-bound proteins were detected with GST, His, MAP4, and Ig antibodies. Band intensities were quantified and plotted against protein concentrations.