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. 2018 Dec 27;142(1):176–192. doi: 10.1093/brain/awy305

Figure 3.

Figure 3

eIF4B phosphorylation at S422 is involved in MMP13-mediated BACE1 regulation. (A) BACE1 protein levels in SH-SY5Y cells treated with 5 μM CL82198 (CL) for 48 h in the absence (CTRL) or presence of 25 μM 4EGI1 (a competitive inhibitor of eIF4E/eIF4G). (B) Dose-response of phosphorylated eIF4B (S422, p-eIF4B) in SH-SY5Y cells treated with 0.1, 1, 2.5, 5 and 10 μM CL82198 for 48 h. (C) p-eIF4B levels in HEK293 cells transfected with either control or MMP13 vector for 48 h in the absence or presence of LY294002 (LY, 5 μM). (D) p-eIF4B levels in HEK293 cells transfected with shRNA control (CTRL) or Mmp13 shRNA-1 vector (shMMP13-1). (E) BACE1 proteins in HEK293 cells transfected with eIF4B S422R mutant (S422R) or wild-type eIF4B vector (eIF4B) in the absence or presence of 5 μM CL82198 (CL) for 48 h. (F) BACE1 protein levels in HEK293 cells transfected with control vector (MOCK), MMP13 or MMP13 and co-transfected with S422R or eIF4B. (G) BACE1 protein levels in HEK293 cells transfected with truncated human BACE1 with a 5′UTR deletion (−5′UTR) or with the entire human BACE1 sequence containing the 5′UTR (+5′UTR) in the absence or presence of 5 μM CL82198 (CL) for 48 h. (H) BACE1 protein levels in HEK293 cells expressing the −5′UTR (left two lanes) or +5′UTR construct (right two lanes) in which eIF4B or S422R was co-transfected. S422R decreased BACE1 protein levels in only the +5′UTR transfected cells. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA, n = 3 or 4).