Figure 4. ILEI Binds to the Cytokine Receptor LIFR.

(a) Mammosphere assay of NMuMG cells stably transduced with either scramble control or LIFR knock-down shRNA (shLIFR) in the presence and absence of 9-day TGFβ pre-treatment (5ng/mL) (error bars represent mean +/− SD; n=5; ****p<0.0001, unpaired Student’s t-test). (b) Quantification of mammosphere formation and self-renewal in E1KD cells stably transduced with either scramble control shRNA or LIFR knock-down (error bars represent mean +/− SD; n=5; p<0.0001 for all passages, paired Student’s t-tests between correlative passage numbers). (c) Quantification of mammosphere formation in E1KD siSCR/ siILEI/ siLIFR cells in the presence and absence of 10nM purified recombinant ILEI (error bars represent mean +/− SD; n=5; **p<0.01, ***p<0.001, ****p<0.0001, unpaired Student’s t-test). (d) BS3 cross-linking in HEK293 cells in the presence or absence of 625pM ¹²⁵I-ILEI and ¹²⁵I-LIF. Radiolabeled BSA was used in control lanes. (e) Cold-competition assay of radiolabeled ¹²⁵I-ILEI with or without 50x cold recombinant ILEI. Indicated samples were BS3 cross-linked to the surface of HEK293 cells and separated by SDS-PAGE. (f) Immunoprecipitation of LIFR in HEK293 cells in the presence and absence of 10nM ILEI followed by immunoblot for either ILEI or LIFR. All lanes were crosslinked with BS3. (g) Overexpression of full-length FLAG-tagged LIFR or vector control followed by ¹²⁵I-LIF (625pM) and ¹²⁵I-ILEI (100pM-2nM) stimulation with BS3 crosslinking. Cell lysates were immunoprecipitated with Flag antibody and separated on an 8% SDS-PAGE gel.