Figure S2.
Data supportingFig. 1—the effects of PTEN on CMA-sensitive proteins are rescued by inhibiting lysosomal proteolysis or by knocking down Lamp2a. (A) Western blots of lysates from AML12 hepatocytes were transduced with either an empty lentiviral vector or one encoding PTEN, and then lysosomal proteolysis was inhibited with 100 μM leupeptin and 20 mM ammonium chloride. CMA-sensitive proteins ACACA, ACLY, FASN, EEF1B2, EEF1D, and EEF2 were assessed for changes. (B) Quantification of blots in A, adjusted to H3 loading control; n = 8 for each group. (C) Western blots of lysates from AML12 hepatocytes that were chemically transfected with negative control siRNA or siRNA targeting Lamp2a or Tsg101 and then transduced with either an empty lentiviral vector or one encoding PTEN. (D) Quantifications of protein levels from C, adjusted to H3 loading control; n = 8 for each group. (E) Western blots of lysates from AML12 hepatocytes that were chemically transfected at ∼50% confluence with negative control siRNA or siRNA targeting Lamp2a or Tsg101, and then harvested at day 2, 3, or 4 after transfection. (F) Quantification of LAMP2A blot in E, adjusted to H3 loading control; n = 7 for each group. (G) Western blots of lysates from AML12 hepatocytes were transduced at ∼50% confluence with either an empty lentiviral vector or one encoding PTEN and then harvested at day 2, 3, or 4 after transfection. (H) Quantification of LAMP2A blot from G, adjusted to H3 loading control. (I) Western blots of lysates from AML12 hepatocytes were treated with 5 μM buparlisib at 50% confluence and harvested at time points of 0, 24, and 48 h. (J) Quantification of LAMP2A blot from I, adjusted to H3 loading control; n = 8 for each group. Bars on graphs show the mean. Error bars are SEM. P values shown on graphs in B and D are from two-tailed unpaired t tests. “PInt” shown in F, H, and J is the interaction term P value from a “full model” two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData FS2.

Data supporting Fig. 1 —the effects of PTEN on CMA-sensitive proteins are rescued by inhibiting lysosomal proteolysis or by knocking down Lamp2a. (A) Western blots of lysates from AML12 hepatocytes were transduced with either an empty lentiviral vector or one encoding PTEN, and then lysosomal proteolysis was inhibited with 100 μM leupeptin and 20 mM ammonium chloride. CMA-sensitive proteins ACACA, ACLY, FASN, EEF1B2, EEF1D, and EEF2 were assessed for changes. (B) Quantification of blots in A, adjusted to H3 loading control; n = 8 for each group. (C) Western blots of lysates from AML12 hepatocytes that were chemically transfected with negative control siRNA or siRNA targeting Lamp2a or Tsg101 and then transduced with either an empty lentiviral vector or one encoding PTEN. (D) Quantifications of protein levels from C, adjusted to H3 loading control; n = 8 for each group. (E) Western blots of lysates from AML12 hepatocytes that were chemically transfected at ∼50% confluence with negative control siRNA or siRNA targeting Lamp2a or Tsg101, and then harvested at day 2, 3, or 4 after transfection. (F) Quantification of LAMP2A blot in E, adjusted to H3 loading control; n = 7 for each group. (G) Western blots of lysates from AML12 hepatocytes were transduced at ∼50% confluence with either an empty lentiviral vector or one encoding PTEN and then harvested at day 2, 3, or 4 after transfection. (H) Quantification of LAMP2A blot from G, adjusted to H3 loading control. (I) Western blots of lysates from AML12 hepatocytes were treated with 5 μM buparlisib at 50% confluence and harvested at time points of 0, 24, and 48 h. (J) Quantification of LAMP2A blot from I, adjusted to H3 loading control; n = 8 for each group. Bars on graphs show the mean. Error bars are SEM. P values shown on graphs in B and D are from two-tailed unpaired t tests. “PInt” shown in F, H, and J is the interaction term P value from a “full model” two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData FS2.

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