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. 2020 May 13;39(13):e103695. doi: 10.15252/embj.2019103695

Figure EV4. Regulation of ADAM17 by PP2A‐B56.

Figure EV4

  1. ITC measurements of the binding of B56α to the indicated ADAM17 peptides.
  2. In vitro phosphorylation of GST and GST‐ADAM17 with PKA.
  3. Western blot of ADAM17 expression in the parental wild‐type (wt) DLD‐1 cell line and ADAM17 knockout (ADAM17−/−) clones #1 and #2.
  4. Sanger sequences of the edited coding region in ADAM17 of DLD‐1 wt and ADAM17−/− clones #1 and #2.
  5. Re‐expression of ADAM17 in ADAM17−/− (clone #1) cells, determined by Western blot.
  6. Amphiregulin (AREG) shedding measured by ELISA of conditioned media from ADAM17−/− cells (clone #1) with or without ADAM17 re‐expression (A17−/−+A17). Two‐sided, unpaired Student's t‐test was applied to test for significant differences **P < 0.01. Mean and standard deviation indicated from three independent experiments.
  7. Western blot of ADAM17 in streptavidin pull‐downs (PD: Strep) and total cell lysates (TCL) from cell surface biotinylated DLD‐1 ADAM17−/− cells (clone #1) re‐expressing ADAM17 variants (wt, I761 or LEE).
  8. Amphiregulin (AREG) shedding measured by ELISA in conditioned media from untreated, H2O2 treated, or radiated DLD‐1 ADAM17−/− cells (clone #2) re‐expressing ADAM17 variants (wt, I761 or LEE). Two‐sided, unpaired Student's t‐test was applied to test for significant differences *P < 0.05, **P < 0.01 and ***P < 0.001. Mean and standard deviation indicated from at least three independent experiments.
  9. Western blot of ADAM17 expression in the parental 4T1 cells line (wt) and 4T1 ADAM17 knockout clone (A17−/−).
  10. Sanger sequences of the edited coding region in Adam17 of the 4T1 wt and A17−/− cell lines. GAPDH was used as an internal loading control in all Western blots. Shedding data represent average values ± SEM of at least 3 independent experiments.

Source data are available online for this figure.