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. 2011 Jan 24;286(12):10367–10377. doi: 10.1074/jbc.M110.206532

FIGURE 8.

FIGURE 8.

MAP1S recruits LC3 to microtubules in an isoform-dependent mode. A, MAP1S recruits LC3 to stabilized microtubules. Association of LC3 (green) with microtubules was monitored in HeLa cells expressing recombinant GFP-LC3 and microtubule stabilizer HA-RASSF1A (red) in the presence or absence of MAP1SSC introduced by transient transfection. A control without cMYC vector (None) was included to test the effect of the cMYC vector. Transfection efficiency driven by the CMYC vector was monitored separately by immunostaining. Yellow indicates overlap of microtubules decorated with RASSF1A and LC3. Images in the fourth panels were captured from the boxed area in the bottom panel that was then subjected to photobleaching under the red laser to demonstrate that the GFP-LC3-associated fibril structures were not caused by leaking through of the red fluorescent signal. B, percentage of cells exhibiting microtubular association of GFP-LC3 in cells expressing both HA-RASSF1A and GFP-LC3 was determined by direct count. Data are the mean ± S.D. of 3 transfection experiments with 100 randomly selected transfected cells including both interphase and mitotic cells counted for each transfection. The p value was determined by Student's t test. C, MAP1S recruits LC3 to microtubules in mitotic cells. Mitotic cells blocked in premetaphase due to RASSF1A were located morphologically from cultures in A. Fibril structures were confirmed to be mitotic spindles made of microtubules by us and others (47, 48). D, a specific profile of interactions among isoforms of MAP1S, LC3I, LC3II, and microtubules is shown. Pelleted microtubules (P) were resuspended in an equal volume of reaction buffer to the supernatants (S). Both fractions were analyzed with antibodies against MAP1S (4G1) and LC3. Tubulin was visualized with Coomassie Blue. Input, reaction mixture in the absence of MAP1S lysates before microtubule assembly; C, control mixture, the same as Input but after assembly and separation of microtubules. A representative of at least three repeats is shown here.