Fig. 2
From: MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP
MANF associates with the ER chaperone BiP in vivo and in vitro. a Coomassie-stained (CBB) SDS-PAGE gel of lysates (Input) and FLAG-M1-immunoaffinity purified proteins (FLAG-IP) from parental CHO-K1 S21 MANF-/- cells and cells stably expressing FLAG-M1-MANF. Indicated bands (1–3) of proteins recovered in complex with FLAG-M1-MANF were individually excised and analyzed by mass spectrometry. The heavy (one asterisk) and light (two asterisks) chains from the ANTI-FLAG M1 agarose affinity gel are indicated. Data representative of two independent experiments are shown. Note that BiP (72 kDa) was unambiguously identified in band 3. b Schema of the domain structure of Chinese hamster BiP (haBiP) and mouse MANF (mMANF). c Bio-layer interferometry (BLI) signals of streptavidin biosensors loaded with biotinylated MANF, the isolated SAP or SAPLIP domains exposed to BiPT229A-V461F (46 µM) in the presence of 2 mM ADP or ATP. Note that SAPLIP does not bind BiP, while MANF and SAP preferentially interact with BiP in its ADP state. d BLI signals of streptavidin biosensors loaded with biotinylated MANF or the isolated SAP domain sequentially exposed to BiP SBD or NBD in presence of ATP or ADP (the trace of the biosensor loaded with SAPLIP is shown in Supplementary Fig. 2a). e Steady-state analysis for the binding affinity of BiP NBD to MANF or the isolated SAP domain. Streptavidin biosensors were loaded with biotinylated BiP NBD and exposed to MANF or SAP at the indicated concentrations. BLI signals at equilibrium were plotted against the concentrations and fitted. Data points represent mean values and SD bars from three independent experiments. Representative raw data traces are shown in Supplementary Fig. 2b, c
