Journal: The FASEB Journal

Article Title: The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction

doi: 10.1096/fj.201802264R

Figure Lengend Snippet: SET interacts with specific PP2A holoenzyme. A–C) Association between endogenous SET and PP2AC in shPP2AA⍺ and shPP2AAβ (A) knockdown cells, interaction between endogenous SET and PP2AC in shB56γ and shB56δ (B) knockdown cells, and association between endogenous SET and PP2AC in shPP2AC⍺ and shPP2ACβ (C) knockdown cells was assessed by PLA. Data are means ± sem of n = 3 independent experiments, analyzed by Student’s t test. *P < 0.05, **P < 0.01. D) Western blot showing successful reconstitution of expression for V5-PP2AA⍺ and V5-PP2AAβ. E) Interaction between endogenous SET and PP2AC in shPP2AA⍺ and shPP2AAβ stable knockdown cells with rescued expression (V5-PP2AA⍺ and V5-PP2AAβ was assessed by PLA using antibodies against SET and PP2AC). Quantitation of PLA. Data are means ± sem of n = 3 independent experiments, analyzed by Student’s t test. ***P < 0.001, ****P < 0.0001. F) Association between endogenous SET and V5-PP2AA⍺ and V5-PP2Aaβ in shPP2AA⍺ and shPP2AAβ stable knockdown cells using antibodies against SET and V5. Quantitation of PLA. Data are means ± sem of n = 3 independent experiments, analyzed by Student’s t test. ****P < 0.0001. G) Western blot showing successful reconstitution of expression for V5-B56γ and V5-B56δ. H) Interaction between endogenous SET and PP2AC in shB56γ and shB56δ stable knockdown cells with rescued expression (V5-B56γ and V5-B56δ) was assessed by PLA using antibodies against SET and PP2AC. Quantitation of PLA. Data are means ± sem of n = 3 independent experiments, analyzed by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. I) Western blot showing successful reconstitution of expression for HA-PP2AC⍺ and HA-PP2ACβ. J) Levels of mRNA showing knockdown of PP2AC⍺ and PP2ACβ obtained by quantitative RT-PCR. K) Association between endogenous SET and HA-tagged PP2AC in shPP2AC⍺ and shPP2ACβ stable knockdown cells with rescued expression (shPP2AC⍺ + HA-PP2AC⍺ and shPP2ACβ + HA-PP2Acβ) was assessed by PLA using antibodies against SET and HA epitope. Quantitation of PLA. Data are means ± sem of n = 3 independent experiments, analyzed by Student’s t test. ****P < 0.0001.

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDF membrane, and probed with the following antibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56γ (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56 γ (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), β-Actin (2066; MilliporeSigma), p-PP2AC Tyr 307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Knockdown, Western Blot, Expressing, Quantitation Assay, Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction

doi: 10.1096/fj.201802264R

Figure Lengend Snippet: Reconstitution of PP2ACα and B56γ expression restores cellular response to FTY720. A) Interaction between endogenous SET and B56γ ± 5 μM FTY720, 5 μM d-e-C18 ceramide, or 1 μM OP449 treatment for 3 h assessed by PLA using antibodies against SET and B56γ. Quantitation of PLA experiments. Error bars are sem of n ≥3 independent experiments per group. Data are means ± sem of n = 3 independent experiments per group, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05. B) Association between endogenous SET and PP2AC X ± 5 μM FTY720, 5 μM d-e-C18 ceramide, or 1 μM OP449 treatment for 3 h assessed by PLA using antibodies against SET and PP2AC. Quantitation of PLA experiments. Error bars are sem of n ≥3 independent experiments per group. Data are means ± sem of n ≥3 independent experiments per group, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. C) Pull-down assay with either GST-tagged PP2A core enzyme (AβCα or AαCα) and SET. D) Pull-down assay with either GST-tagged PP2A core enzyme (AβCα) and SET with increasing (0–50 μM) FTY720. E) Pull-down assay with either GST-tagged PP2A holoenzyme (AβCαBγ1) and SET with increasing (0–100 μM) FTY720. F) Pull-down assay with either GST-tagged Bγ1 and SET with increasing (0–50 μM) FTY720. G) SET oligomerization is essential for PP2A inhibition. Lipid binding prevents SET oligomerization resulting in active PP2A. Additionally, SET remains with FTY720-activated PP2A by maintaining contact with the B56γ subunit as a possible regulatory mechanism.

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDF membrane, and probed with the following antibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56γ (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56 γ (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), β-Actin (2066; MilliporeSigma), p-PP2AC Tyr 307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Expressing, Quantitation Assay, Pull Down Assay, Inhibition, Binding Assay

Journal: The FASEB Journal

Article Title: The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction

doi: 10.1096/fj.201802264R

Figure Lengend Snippet: Phosphorylation of SET influences PP2A activity. A) Association between endogenous SET, FLAG-SETWT, FLAG-SETS171A, and FLAG-SETS171E, mutants, and PP2AC ± 5 μM FTY720 with a pCDH empty vector control measured by PLA using antibodies against SET, FLAG, and PP2AC. Data are means ± sem of n = 3 independent experiments, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001. B) Association between FLAG-SETS171A and FLAG-SETS171E and B56γ ± 5 μM FTY720 assessed by PLA using antibodies against FLAG and B56γ. Data are means ± sem of n = 3 independent experiments analyzed by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01. C) Western blot of PP2A-p-Tyr307 of PP2AC as a marker of activity normalized to β-actin and FLAG expression. Representative blot of n = 3 individual experiments. D) Blots were quantified with ImageJ. Data are means normalized to actin and FLAG ± sd and analyzed by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05. E) Quantification of GA cross-linking of FLAG-SETWT, FLAG-SETS171A, and FLAG-SETS171E oligomers in response to 3-h treatment with 5 μM FTY720. Data are means ± sd (n = 3 independent experiments) analyzed by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05.

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDF membrane, and probed with the following antibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56γ (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56 γ (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), β-Actin (2066; MilliporeSigma), p-PP2AC Tyr 307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Phospho-proteomics, Activity Assay, Plasmid Preparation, Control, Western Blot, Marker, Expressing