Figure S4. Identifying TDP-43... | Rockefeller University Press

Figure S4.

$Identifying TDP-43 interactome and mapping the binding site of TDP-43 for translational factors. (A) Schematic overview of subcellular fraction, affinity purification, and MS strategies used in this study. After the removal of mitochondrial, nuclear, and unbroken cells, the supernatant was subjected to affinity purification of Strep-tagged TDP-43 by Strep-Tactin beads. The purified proteins were digested, labeled by stable isotopes, and finally analyzed by quantitative MS. HEK293 cells with the knockout (KO) of TDP-43 were generated using CRISPR/Cas9 genome editing and used to express Strep-tagged TDP-43WT or TDP-43ΔCR by transient transfection. TDP-43 KO cells expressing TDP-43WT or TDP-43ΔCR without Strep tag were used as a control for IP. m/z, mass-to-charge ratio. (B) Representative immunoblot of coIP between exogenously expressed Strep-Flag–tagged PABPC4/PABPC1 and GFP-tagged TDP-43ΔCR in HEK293 cells. Cells transfected with empty vector were also included as controls. (C) Representative immunoblot of coIP between exogenously expressed GFP-tagged EEF1A1 and Strep-Flag–tagged TDP-43WT or Strep-Flag–tagged TDP-43ΔCR in HEK293 cells. Cells transfected with empty vector were also included as controls. (D) Representative immunoblot of coIP between exogenously expressed GFP-PABPC1 and Strep-Flag–tagged TDP-43WT or Strep-Flag–tagged TDP-43ΔCR in HEK293 cells. (E) Representative immunoblot of coIP between exogenously expressed GFP-tagged PARP1 and Strep-Flag–tagged TDP-43WT or Strep-Flag–tagged TDP-43ΔCR in HEK293 cells. (F) Schematic representation of TDP-43 deletion mutants used for mapping the binding site of TDP-43 for translational factors. (G–I) Representative immunoblot of coIP between transfected exogenously expressed GFP-tagged PABPC4 and the indicated Strep-Flag–tagged TDP-43ΔCR deletion mutants in HEK293 cells. (J) Representative immunoblot of coIP between exogenously expressed Strep-Flag–tagged TDP-43 WT or Δ99-105 and endogenous PABPC4, RPS6, and RPL7 in HEK293 cells. (K) Representative immunoblot of TDP-43 and mutations in exogenously expressed Flag-tagged TDP-43, Flag-tagged TDP-43ΔCR, and Flag-tagged TDP-43ΔCR/Δ99-105 in TDP-43 KO cells.$

Identifying TDP-43 interactome and mapping the binding site of TDP-43 for translational factors. (A) Schematic overview of subcellular fraction, affinity purification, and MS strategies used in this study. After the removal of mitochondrial, nuclear, and unbroken cells, the supernatant was subjected to affinity purification of Strep-tagged TDP-43 by Strep-Tactin beads. The purified proteins were digested, labeled by stable isotopes, and finally analyzed by quantitative MS. HEK293 cells with the knockout (KO) of TDP-43 were generated using CRISPR/Cas9 genome editing and used to express Strep-tagged TDP-43WT or TDP-43ΔCR by transient transfection. TDP-43 KO cells expressing TDP-43WT or TDP-43ΔCR without Strep tag were used as a control for IP. m/z, mass-to-charge ratio. (B) Representative immunoblot of coIP between exogenously expressed Strep-Flag–tagged PABPC4/PABPC1 and GFP-tagged TDP-43ΔCR in HEK293 cells. Cells transfected with empty vector were also included as controls. (C) Representative immunoblot of coIP between exogenously expressed GFP-tagged EEF1A1 and Strep-Flag–tagged TDP-43WT or Strep-Flag–tagged TDP-43ΔCR in HEK293 cells. Cells transfected with empty vector were also included as controls. (D) Representative immunoblot of coIP between exogenously expressed GFP-PABPC1 and Strep-Flag–tagged TDP-43WT or Strep-Flag–tagged TDP-43ΔCR in HEK293 cells. (E) Representative immunoblot of coIP between exogenously expressed GFP-tagged PARP1 and Strep-Flag–tagged TDP-43WT or Strep-Flag–tagged TDP-43ΔCR in HEK293 cells. (F) Schematic representation of TDP-43 deletion mutants used for mapping the binding site of TDP-43 for translational factors. (G–I) Representative immunoblot of coIP between transfected exogenously expressed GFP-tagged PABPC4 and the indicated Strep-Flag–tagged TDP-43ΔCR deletion mutants in HEK293 cells. (J) Representative immunoblot of coIP between exogenously expressed Strep-Flag–tagged TDP-43 WT or Δ99-105 and endogenous PABPC4, RPS6, and RPL7 in HEK293 cells. (K) Representative immunoblot of TDP-43 and mutations in exogenously expressed Flag-tagged TDP-43, Flag-tagged TDP-43ΔCR, and Flag-tagged TDP-43ΔCR/Δ99-105 in TDP-43 KO cells.

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