Fig. 1
From: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
A heightened UPR in MANF knockout cells. a Schematic illustration of the CHO-K1 MANF gene. The encoded N-terminal SAPLIP (Saposin-like protein; blue) and the C-terminal SAP (SAF-A/B, Acinus, and PIAS; red) domains as well as the signal peptide (SP), linker region (black), and RTDL motif are shown. The encoded amino acid sequence surrounding the mutations (caused by CRISPR-Cas9-mediated nucleotide insertion or deletion) are noted for each allele. Both mutations result in premature termination of translation interrupting the SAPLIP domain and deleting the SAP domain. b Immunoblot of MANF in lysates of CHO-K1 S21 wild-type (wt) and MANF−/− cells. The BiP and actin signal in the samples is also shown. This experiment has been reproduced independently three times. Note that no MANF signal was detected in the sample from MANF-/- cells lysate. c Flow cytometry plots of CHOP:GFP and XBP1s:Turquoise UPR reporters in untreated and thapsigargin-treated (16 h) CHO-K1 S21 wild-type and MANF-/- cells. The inset shows the median ± SD of the GFP and Turquoise fluorescence signals of the wt (red) and MANF-/- (blue) cells from three independent experiments (similar results were obtained with two independently derived MANF-/- clones). d Immunoblot of FLAG-tagged MANF from cell lysates and FLAG-M1-immunoaffinity purified proteins (FLAG IP) from the corresponding cell culture supernatants (media) of parental CHO-K1 S21 MANF-/- cells and cells stably expressing FLAG-M1-MANF. Cells were untreated or treated with thapsigargin (Tg; 0.5 µM) for the indicated times. The content of BiP and actin in the samples is provided as a loading control. Uncropped images for panels (b) and (d) and source data for panel (c) are provided as a Source Data file
