1. Fig. 6 The C9ORF72/SMCR8 complex regulates ULK1.
The C9ORF72/SMCR8 complex regulates ULK1. (A) The interaction between C9ORF72/SMCR8 complex with ULK1 is enhanced under starvation conditions. Flag-C9ORF72 and RFP-SMCR8 were transfected into HEK293 cells with or without amino acid starvation for 1 hour before protein lysate collection. C9ORF72 protein was immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analyses using antibodies as listed. (B) Western blot analysis of Ulk1 expression in wild-type or Smcr8 mutant MEFs. β-Actin serves as the loading control. (C) Quantification of Ulk1 expression relative to actin. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). (D) Ulk1 protein in wild-type or Smcr8 mutant MEFs was immunoprecipitated with Ulk1 antibodies followed by Western blot analyses. IgG, immunoglobulin G. (E) Quantification of relative Ulk1 and Atg13 protein levels. Error bars represent SEM of three measurements from three independent experiments; *P < 0.01 (Student’s t test). (F) Western blot analysis of phospho-Ulk1 (p-Ser757) expression. Wild-type or Smcr8 mutant MEFs were treated with DMSO or rapamycin (0.1 μM) for 3.5 hours before protein lysate collection. (G) Quantification of phospo-Ulk1 (p-Ulk1)/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). (H) Western blot analysis of phospho-Ulk1 (p-Ser757) expression. Wild-type or Smcr8 mutant MEFs were infected with lentiviruses expressing control or C9orf72 shRNA constructs. β-Actin serves as the loading control. (I) Quantification of relative levels of phospho-Ulk1. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test).
Mei Yang et al. Sci Adv 2016;2:e
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