Fig. 6
From: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
MANF stabilizes high-molecular-weight BiP complexes in stressed cells. a Coomassie-stained (CBB) SDS-PAGE gel and corresponding BiP immunoblot of the cell lysate (Input) and detergent insoluble high-molecular-weight (HMW) complexes from untreated and tunicamycin-treated (Tm; 2.5 μg/mL, 15 h) CHO-K1 S21 wild-type (wt) and MANF-/- cells. Shown is a representative of three experiments. b Schema of the design of the SILAC experiment to quantify relative changes in abundance of BiP peptides incorporated into detergent-insoluble high-molecular-weight complexes in CHO-K1 S21 wild-type and MANF-/- cells treated with tunicamycin (Tm; 2.5 μg/mL, 15 h). c LC-MS spectra of a representative doubly charged tryptic BiP peptide (VEIIANDQGNR60) from the input (top) and HMW complexes (bottom) of experiments as outlined in (b). The spectrum on the left is from lysate of MANF-/- cells cultured in light medium combined with lysate of wild-type cells cultured in heavy medium, and the spectrum on the right is from lysate of MANF-/- cells cultured in heavy medium combined with lysate of wild-type cells cultured in light medium. d Averaged normalized ratios (Rnrl) of BiP peptides identified in the LC-MS spectra from the MANF-/- cells versus wild-type cells in the input and HMW complexes fraction from the two experiments as described in (c). The position of the peptides on the BiP sequence (654 amino acids) is indicated by the brackets. The BiP signal peptide (SP), nucleotide-binding domain (NBD), and substrate-binding domain (SBD) are indicated. Uncropped image for panel (a) and source data for panel (d) are provided as a Source Data file
