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. 2019 Aug 5;33(11):12019–12035. doi: 10.1096/fj.201802825RR

Figure 1.

Figure 1

GULP1 is a phosphoprotein, and GULP1 T35 phosphomimetic mutation reduces GULP1-mediated APP processing. A) HEK293, CHO, hamster brain, and GULP1-transfected CHO cell lysates were fractionated by a high-resolution NuPAGE Bis-Tris Precast Gel and then subjected to Western blot analysis using a rat anti-GULP1 antibody. Two closely migrating GULP1 bands were detected by the anti-GULP1 antibody in all the samples (arrowed). Transfected GULP1 was used as a size control. Two exposures of the same blot are shown. The amounts of protein lysates in each lane are indicated. B–D) Untransfected (UT) HA-GULP1–transfected CHO (B), UT CHO cells (C), and rat brain (D) were harvested in 1× dephosphorylation buffer. The protein lysates were incubated at 30°C either with (+) or without (−) λ-protein phosphatase for 60 min. The samples were then analyzed by NuPAGE Bis-Tris Precast Gels and Western blotting. Transfected HA-GULP1 (B) and endogenous GULP1 (C, D) were detected by a mouse anti-HA 12CA5 (Roche) and a rat anti-GULP1, respectively. The slow-migrating GULP1 bands (arrows) (B–D) disappeared after λ-protein phosphatase treatment. E) CHO cells were cotransfected with APP-GAL4, pFR-Luc, and phRL-TK together with either empty vector control plasmid (mock) or different phosphomimetic mutants of GULP1. GULP1 T35 mutant shows reduced ability to stimulate GAL4-APP cleavage as compared with GULP1 WT; n = 5. Results are means ± sd. *P < 0.001 compared with GULP1 transfected cells. F) Alignment of GULP1 sequence around T35 (bolded; reference to human) from various species. G) Immunoblot analysis of APP CTFs from CHO cells transfected with APP, APP + BACE1, APP + BACE1 + GULP1, APP + BACE1 + GULP1 T35D, or APP + BACE1 + GULP1 T35A (top panel). The expression of transfected APP, BACE1, and GULP1 were also determined. The amounts of APP CTF-α, -β′ and -β′ were quantified. Full-length APP and APP CTFs were detected by a rabbit anti-APP. HA-GULP1 and BACE1-myc were probed by a mouse anti-HA (12CA5) and a mouse anti-myc (9B11), respectively. Data for graphs were obtained from 3 independent experiments. Ns, not significant (n = 3). Results are means ± sd. *P < 0.001, **P < 0.01. H) CHO cells were cotransfected with APP either mock, GULP1, GULP1 T35A, or GULP1 T35D. Forty-eight hours post-transfection, cell culture medium was aspirated and changed to fresh medium. The level of secreted Aβx-40 was assayed using an ELISA kit 7 h after the change of medium (n = 5). *P < 0.001 compared with GULP1 transfected cells. The effect of GULP1 on Aβx-40 liberation is reduced by GULP1 T35 phosphomimetic mutation.