Figure 2
From: Monitoring LC3- or GABARAP-positive autophagic membranes using modified RavZ-based probes
Roles of the LIR motifs of RavZ on mATG8-positive autophagic membrane targeting. (A,B) Contribution of the LIR motifs of GFP-fused LIR motifs of RavZ in autophagosome targeting. RavZ(ΔCA)mLIR1/2-3-GFP, an LIR1/2/3 mutant of RavZ(ΔCA)-GFP; LIR(1/2-3)-GFP, an MT domain deletion mutant of RavZ(ΔCA)-GFP. Confocal images (A) depicting the cellular localization of GFP-fused LIR motifs of RavZ in rapamycin (Rapa)/NH4Cl-treated MEF cells. Scale bar: 10 μm. The bar graphs (B) illustrate the GFP fluorescence intensities of the autophagic membranes and the cytosol (the A/C ratio) (n = 75 for each group). ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). (C,D) mATG8 protein-binding properties of the GFP-fused LIR motifs of RavZ proteins using GST-pulldown assays and quantification analysis for the binding. Extended blot images including these data are presented in Supplementary Fig. 6A. The bar graphs (D) illustrate relative quantification of the level of bound GFP-constructs in GST-pulldown assay. The levels of bound GFP-constructs intensity were normalized to the intensity of the expressed GFP-constructs (input). The data are presented as the mean ± SEM of three independent experiments. RAP, RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2.
