Vinblas tine is able to depolymerize both acetylated and non acetylated microtubules, but enhances tubulin acet ylation. The autophagic responses to the treatments of different microtubular interfering agents reveal that reg ular non acetylated microtubules regulate the efficiency of but are not essential for the conversion of LC3I to LC3II. Acetylated microtubules sellckchem are required for LC3II degradation. Methods Reagents and antibodies Microtubule interfering reagents paclitaxel, nocodazole and vinblastine sulfate salt, and lysosomal inhibitor bafi lomycin A1 and ammonium chloride were purchased from Sigma. Monoclonal antibodies against b actin, acetylated tubulin and LAMP2, polyclonal antibody against b tubulin, and FITC and Rhodamine conjugated secondary anti bodies were purchased from Santa Cruz Biotechnology, Inc.
Polyclonal antibodies against LC3 and P62 were from Nuvus Bio and ENZO Life Science, respectively. Immunoblot analysis Unless otherwise indicated, lysates were prepared in lysis buffer from cells treated with different drugs overnight, specific times and cell fractions enriched for mitotic or interphase cells as described. Immunoblots were prepared from equal amounts of samples separated on SDS PAGE and analyzed with the indicated antibodies. b actin served as loading control. Protein band profiles were detected with the Amersham ECL Plus detection system and a series of images with different exposure times were archived. Data presented in the text for immunoblot or immunohistochemical analysis were representatives of at least three independent experi ments.
Some bands necessarily appear overexposed because of attempts to display the weakest band. Rela tive intensities of bands were calculated using ImageJ from scanned images of the respective immunoblot in the linear range and adjusted based on the respective b actin intensity. The intensity of bands in controls was assigned a unit of 1. Immunofluorescence analysis A stable HeLa cell line expressing GFP LC3 fusion pro tein was established as described As described, we established a stable HeLa cell line expressing GFP fusion of a mutant version of LC3 that carries a deletion of the 22 amino acid residues of LC3 C terminus and has the lipid conjugation site Gly cine at residue number 120 mutated into Alanine so that it exhibits defective in autophagy initiation.
Spread mono layered interphase cells and round mitotic cells were visualized with a Zeiss LSM510 laser confocal system. GFP LC3 labeled autophagosomes, MitoTracker Red CMXRos labeled mitochondria, pri mary antibody and corresponding FITC or Rhodamine conjugated Carfilzomib secondary antibody were used to visualize microtubules and lysosomes. We specifically demonstrated the relationship between chromosomes and GFP LC3 previously.