Targeting the Unfolded Protein Response in Glioblastoma Cells with the Fusion Protein EGF-SubA
Figure 2
SubA and EGF-SubA cleaves GRP78 and activates the UPR.
Exponentially growing glioblastoma cell lines and normal human astrocytes (NHA) were (A) treated with SubA or EGF-SubA at the specified picomolar concentrations for 24 h or (B) exposed EGF-SubA (1 pM) for the specified time periods. Total cellular protein was isolated and immunoblotting was performed with anti-GRP78 antibody. SubA and EGF-SubA cleaved the endogenous GRP78 (78 kDa) resulting in an additional smaller fragment of 28 kDa (cGRP78). (C-E) Total cellular protein and RNA were isolated from U251 cells exposed to EGF-SubA at the stated concentrations for 24 h. EGF-SubA induced GRP78 cleavage resulted in nuclear localization of ATF6 (C; nATF6), a dose-dependent phosphorylation of PERK (D; pPERK), and Ire1 activation, determined by Xbp1 mRNA splicing (E). Each figure is a representative of three independent experiments.