Figure 4. Phosphorylation of TEX264 LIR increases its affinity to ATG8s.

• A
  Binding affinity of TEX264 LIR peptides (TEX264^269–278) to LC3 and GABARAP was measured by ITC (n = 1). The error bars show the fitting error.
• B
  HEK293T cells transiently expressing WT or mutated TEX264‐FLAG were subjected to immunoprecipitation with an anti‐FLAG antibody and detected with the indicated antibodies.
• C, D
  MEFs stably expressing TEX264‐GFP or its mutants were cultured in starvation media and immunostained with an anti‐LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264‐GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25^th–75^th percentile), and whiskers the 0^th–100^th percentile. Differences were statistically analyzed by one‐way ANOVA and Tukey’s multiple comparison test. Data were collected from 70 puncta for each cell type (D).
• E, F
  WT and TEX264‐KO (expressing WT or TEX264‐FLAG mutants) HeLa cells stably expressing the ER‐phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP‐GFP were quantified and the ratio of RFP:RFP‐GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one‐way ANOVA and Tukey’s multiple comparison test (F).