Fig. 6: CCR4-NOT complex associates with LC3B, promoting efficient LMD.
From: LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy
a IPs of endogenous LC3B. The extracts of HEK293T cells either treated or not treated with Rapa + CQ were subjected to IPs using α-LC3B antibody or nonspecific rIgG. The intensities of each western blot image were quantitated. The intensities of coimmunoprecipitated proteins were normalized to those of immunoprecipitated LC3B. The relative levels obtained in untreated cells were arbitrarily set to 1.0. n = 3. b, c IPs of endogenous LC3B upon downregulation of both ATG5 and ATG7. As performed in a, except that the cells were either undepleted or depleted of ATG5 and ATG7. b Western blotting proving specific downregulation of endogenous ATG5 and ATG7. c Western blotting of cellular proteins before or after IPs of LC3B. d The proximity ligation assay (PLA) between endogenous LC3B and either CNOT1 or CNOT7. PLA experiments using the indicated antibodies were performed on HeLa cells treated with either DMSO or Rapa + CQ. The PLA images are shown in Supplementary Fig. 10. The number of PLA spots per cell was quantified and is presented in this panel. Box-whiskers show maximum, third quartile to first quartile, median and minimum; n = 767 cells examined over three independent experiments. e, f Effect of CNOT1 or CNOT7 downregulation on LMD. e Western blotting showing specific downregulation of endogenous CNOT1 and CNOT7. f LMD efficiency of RLuc-P3′-WT reporter mRNA, endogenous PRMT1 mRNA, and MARS1 mRNA. n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; *p < 0.05; **p < 0.01 (The exact p values are provided in Source Data file). Source data are provided as a Source Data file.
