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. 2015 Dec 22;113(5):E509–E518. doi: 10.1073/pnas.1512952113

Fig. 4.

Fig. 4.

Activity of γ-secretase after treatment with reducing reagents. (A) Western blot of NICD product after the cleavage of V1711, pSub1711, and Ub1711 with γ-secretase in the presence and absence of DTT. (B) Cleavage of cysteine-free Ub1711 with untreated and reduced γ-secretase (mean ± SD, n = 2). (C) Western blot of NICD product after γ-secretase cleavage of pSub1711 untreated and in the presence of DTT, BME, or TCEP. (D) Native blue gel electrophoresis of γ-secretase untreated or in the presence of DTT or Nonidet P-40 detergent. Nicastrin and presenilin CTFs were detected by Western blot. (E) Kinetic analysis of V1711 cleavage in the presence of DTT (mean ± SD, n = 3). (F) Kinetic analysis of Ub1711 cleavage in the presence and absence of DTT (mean ± SD, n = 3). (G) Cleavage of pSub1711 (n = 6) and N-terminally ubiquitin (Ub)-tagged notch (n = 3), APLP1 (n = 2), APLP2 (n = 2), Navβ (n = 2), and C83 (n = 2) (mean ± SD, t test; P values are shown above each substrate).