Figure 2Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. Data information: the experiments were performed at least three times independently with similar results.Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D) Data information: , the experiments were performed at least three times independently with similar results.Western blot analysis of HEK293T cells transfected with empty Flag vector (-) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 hours (E). Data information: the experiments were performed at least three times independently with similar results.F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 hours. Scale bar, 10 μm. Data information: the experiments were performed at least three times independently with similar results.HA-ULK1 levels in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) Western blots against HA-ULK1 and actin control (G, were subjected to densitometric quantitation (H, Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H , **P < 0.01.endogenous ULK1 levels (I, J) in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells or without TRIM27 RNAi (I, J). and actin control I) were subjected to densitometric quantitation J). Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests J), **P < 0.01.Ubiquitination assays were performed in HEK293T cells after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) in the presence or absence of Flag-TRIM27. Data information: , the experiments were performed at least three times independently with similar results.Ubiquitination assays were performed in HEK293T cells after co-transfection with double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27. Data information: the experiments were performed at least three times independently with similar results.M. Ubiquitination of HA-ULK1 in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively. Data information: the experiments were performed at least three times independently with similar results.N. In vitro ubiquitination assays against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27. Data information: the experiments were performed at least three times independently with similar results.O. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0h) and after treatment with CHX for 12 hours. Data information: the experiments were performed at least three times independently with similar results.