Upon Glc replenishment, BiP ATPase activity is required to solubilize wtFVIII-CMy. (A) BiP binds LMW-FVIII. Lysates from H9 cells treated with or without NaB were subjected to sucrose gradient sedimentation, and FVIII from fractions 5 (light fraction [L]) and 10 (heavy fraction [H]) was immunoprecipitated. After elution, coimmunoprecipitated BiP was analyzed by western blotting with FVIII and BiP antibodies. (i) Western blots for FVIII and BiP are shown for cell lysates. (ii) Western blots are shown for FVIII IP and post-IP supernatants. (B) The figure shows the crystal structure of BiP in the ATP-bound conformation. The white arrow indicates the cleavage site for SubAB protease that connects the nucleotide-binding domain (NBD; green) with the substrate-binding domain (SBD; yellow). The C-terminal lid is indicated in pink. The model is based on PDB-6ASY.⁵⁶  (C) BiP cleavage induces wtFVIII aggregation. H9 cells were treated with 5 μM SAHA for 24 hours and BiP cleavage was initiated by treating cells with 1 or 2 µg/mL SubAB (AB) or mutant SubA₂₇₂B (mt) for the last 2 hours of SAHA treatment. Top, CA membrane filtration of cell lysates shows relative levels of FVIII aggregation compared with cells not treated with SubAB (UT). Bottom, Western blot is shown for cell lysates using indicated antibodies. The abundance of intact BiP (∼75 kDa) was quantified relative to vinculin (loading control) and is represented as a percentage of untreated. (D) BiP is required for disassociation of FVIII aggregates. H9 cells treated with SAHA for 18 hours and then with 2DG and NaN₃ for 2 hours in the absence of Glc. Then, Glc was repleted in the presence of either 2 µg/mL SubAB (AB) or SubA₂₇₂B (mt) or CST (100 and 200 µM) for 4 hours. Cell lysates were filtered through a CA membrane and analyzed by blotting. The percentage of FVIII aggregation was quantified from the FVIII signals on CA relative to western blotting compared with untreated control. C-ter BiP is SubAB cleaved C-terminal BiP. (E) BiP inactivation induces wtFVIII-CMy aggregation. 293T cells expressing wtFVIII-CMy were treated with or without SubAB or mtSubAB (2 µg/mL) for 2.5 hours and then harvested for western blotting and CA membrane filtration. The relative wtFVIII-CMy western blot intensities were normalized to vinculin. CA intensities were normalized to FVIII western blot intensities. Black bars = biological duplicates; blue bars = technical triplicates. These data are a representative experiment with biological duplicates. (F) wtFVIII-CMy interacts with BiP. Cell lysates from 293T cells expressing wtFVIII-CMy and BiP-Flag (BiP-F) or BiP-Flag (V461F) (BiPmt-F), a peptide-binding defective mutant, were used for Flag IP. After IP and blotting, BiP was detected with Flag antibody (top) and FVIII-CMy with Cpep antibody (bottom) to compare untreated cells and cells treated with 2DG and NaN₃ for 2.5 hours. LC and HC represent immunoglobulin light and heavy chains of Flag antibody. UI, uninduced.