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. Author manuscript; available in PMC: 2022 Aug 18.
Published in final edited form as: J Am Chem Soc. 2021 Aug 4;143(32):12675–12687. doi: 10.1021/jacs.1c05386

Figure 4|. NBDY is phosphorylated at S61 downstream of protein kinase C (PKC) during EGFR signaling.

Figure 4|

(a) NBDY phosphorylation site observed during EGFR signaling (red). (b) EGF-dependent NBDY phosphorylation site identified by LC-MS/MS. Red peaks, y-ions; yellow peaks, b-ions. (c) Validation of anti-NBDY pS61 antibody. Extracts were prepared from HEK 293T cells transiently expressing FLAG-NBDY or FLAG-NBDY S61A with or without EGF treatment. λPPase indicates treatment with λ-phosphatase before SDS–PAGE. (d) Phos-tag PAGE and Western blotting validation of NBDY pS61. Transiently expressed FLAG-NBDY was immunopurified from HEK 293T cells with or without EGF treatment. β-Actin, loading control. (e) Endogenous NBDY pS61 was assayed in lung cancer cells with wild-type EGFR (A549), activating EGFR mutation (HCC827 and H3255), or activating EGFR mutation and erlotinib resistance (H1975) in the presence of vehicle or erlotinib. (f) NBDY pS61 is regulated by PKC. Extracts were prepared from FLAG-NBDY expressing cells treated with vehicle, or with EGF in the presence of vehicle, EGFR inhibitor (AG1478) or PKC inhibitor (BIMII, bisindolylmaleimide II) followed by immunopurification and western blotting. (g) In vitro kinase assay. Purified NBDY was incubated with purified control kinases (GRK2, GSK3β) or PKCα in the presence of ATP, then subjected to Western blotting. (h) Model: NBDY is phosphorylated at S61 downstream of PKC during EGFR signaling.