Mutation of R68A impairs the autophagosomal localization and autophagic flux of LC3B protein. A. Representative images of autophagosomal localization of GFP-LC3B and GFP-LC3B-R68A in normal culture condition and starvation conditions. GFP-LC3B and GFP-LC3B-R68A were stably expressed in HeLa cells. After treated with or without starvation for 2 hours, cells were subjected to immunostaining with p62 protein as autophagosome marker. The average punctate number in each cell is shown in the right. More than 100 cells were examined for the GFP puncta number and data were obtained from 3 independent experiments and represented as mean ± SEM. Bar = 20 μm. B. Autophagic flux was determined by monitoring LC3B-II turnover. HeLa cells expressing myc-LC3B or myc-LC3B-R68A were treated with or without 20mM NH4Cl for 4 hours in normal culture condition or starvation condition. Then the cell lysates were subjected to SDS-PAGE and immunobloting with anti-myc and anti-β-actin antibodies. The ratios of LC3B-II in the total LC3B protein according to the intensity of band were then plot on the right. Data were obtained from 3 independent experiments and represented as mean ± S.D. * p < 0.05, ** p < 0.01.
Liu et al. BMC Cell Biology 2013 14:27 doi:10.1186/1471-2121-14-27