Figure 5.

Lack of Hjv interaction decreases the level of membrane-associated Neo1, but not Hjv in the liver. (A) Representative images of western blot analysis for endogenously expressed Neo1, Tfr2, Hjv, and β-actin, as well as the introduced fNeo1 and fNeo1^L1046E, in the liver membrane preparation (250 µg protein) of Neo1^fl/fl;Alb-Cre⁻ mice, PBS-injected Neo1^fl/fl;Alb-Cre⁺ mice (–), and Neo1^fl/fl;Alb-Cre⁺ mice transduced with AAV8-fNeo1 and fNeo1^L1046E. (B) Diagrams of the predicted cleavage sites by α- and γ-secretases in fNeo1, as well as the antibodies used for western blot analysis in panels A and C. (C) Representative images of western blot analysis for concentrated fNeo1 and fNeo1^L1046E from the liver membrane preparation (membrane) and the whole liver extracts (liver extracts) of Neo1^fl/fl;Alb-Cre⁺ mice transduced with AAV8-fNeo1 and fNeo1^L1046E. fNeo1 and fNeo1^L1046E from ∼2 mg extract proteins was pulled down by using anti-FLAG affinity gel (A2220; Sigma), followed by elution using the 3xFLAG peptide at ∼200 µg/mL (F4799; Sigma) and immunodetection using anti-NEO1 FNIII 1-6 and anti-FLAG antibody.