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. 2020 Nov 20;40(2):e104532. doi: 10.15252/embj.2020104532

Figure 7. Peli1 regulates the stability of TSC complex through ubiquitination of TSC1.

Figure 7

  1. Analysis of ubiquitin conjugation to wild‐type (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag‐tagged TSC1 and its mutants along with (+) or without (–) the indicated expression vectors.
  2. Immunoblot analysis of the indicated phosphorylated (p‐) and total proteins in lysates of TSC1‐KO MEFs transfected with (+) or without (–) TSC1 wild‐type or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β‐Actin.
  3. Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (–) the indicated expression vectors.
  4. Co‐immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wild‐type and K30A in TSC1‐KO MEFs transfected (for 48 h) with (+) or without (–) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti‐Flag IP blot in left panel.
  5. Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1‐KO MEFs transfected (for 48 h) with (+) or without (−) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.
  6. Immunoblot analysis of endogenous TSC2 in TSC1‐KO MEFs transfected (for 48 h) with Myc‐TSC1 wild‐type or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.

Data information: Summary graphs in B, D, and F are densitometric quantification of the indicated protein bands based on three independent experiments, and bar graphs are presented as mean ± SEM. P values are determined by a two‐tailed unpaired Student’s t‐test. *P < 0.05.

Source data are available online for this figure.