Fig. 2: Characterization of substrate and product of the iCliPSpy assay.
a E. coli T7 Express cells harboring the plasmids 1, pETDuet-1 (control), 2, pETDuet-1-His-MBP-TNFα-(1-39)-L31P (expressing a substrate for I-CLiPs), 3, pETDuet-1-MBPmut-TNFα-(1-39)-L31P (expressing an alternative substrate for I-CLiPs) or 4, pETDuet-1-MBPmut-TNFα-(1-34)-L31P (expressing the putative product of intramembrane proteolysis), were incubated overnight on an agar plate at 37 °C followed by another day at room temperature. b E. coli T7 Express cells harboring the plasmids 1, pETDuet-1, 2, pETDuet-1-His-MBP-TNFα-(1-39)-L31P, 3, pETDuet-1-MBPmut-TNFα-(1-39)-L31P or 4, pETDuet-1-MBPmut-TNFα-(1-34)-L31P, were grown on an agar plate for six days at room temperature, scraped from the plate, resuspended in TN buffer, and harvested by centrifugation and c then further analyzed by SDS-PAGE (after solubilization by boiling in Laemmli sample buffer). d MBPmut-TNFα-(1-39)-L31P and MBPmut-TNFα-(1-34)-L31P were purified by amylose affinity chromatography from the membrane fraction using DDM as solubilizing detergent (left and middle columns) from IPTG induced E. coli T7 Express cells harboring the above-listed plasmids. Purification of MBPmut-TNFα-(1-34)-L31P was repeated after addition of 25 µM hemin (right column) to membrane proteins solubilized with DDM. Purified MBP fusion proteins were then analyzed by e SDS PAGE and f UV/Vis spectroscopy [plus 0.2% DDM: blue trace, MBPmut-TNFα-(1-39)-L31P; red trace, MBPmut-TNFα-(1-34)-L31P; grey trace, MBPmut-TNFα-(1-34)-L31P plus hemin; minus DDM: yellow trace, MBPmut-TNFα-(1-34)-L31P plus hemin]. When DDM is removed from hemin reconstituted MBPmut-TNFα-(1-34)-L31P by washing the amylose column with buffer containing no DDM, the typical absorbance maximum of about 373 nm for mono-thiolate ligated heme is observed25,56, showing that presence of DDM leads to a bathochromic shift of the absorbance maximum from 373 nm to about 392 nm for mono-thiolate ligated heme. Free hemin in the elution buffer used with 0.2% DDM has an absorbance maximum at 405 nm. In vivo detection of MBP-TNFα fusion proteins, as shown in a, was repeated once (and in a different way in Fig. 3 a and Supplementary Fig. 2a), and one representative result is shown. Detection of MBP-TNFα fusion proteins in cells by SDS-PAGE (b and c) was done once, and the UV/Vis spectra of purified MBPmut-TNFα-(1-39)-L31P and MBPmut-TNFα-(1-34)-L31P fusion proteins (f), were checked in a second experiment.
