Fig. 5.

Cavin-1–SOCS3 interaction requires the SOCS3 SH2 PEST motif. a Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either WT or ΔPEST Flag-SOCS3 were incubated with 100 nM N-terminally biotinylated peptides corresponding to the Tyr759 motif of gp130 in its phosphorylated (pY) or non-phosphorylated (Y) forms and streptavidin–agarose beads prior to loading with whole-cell lysate samples for SDS-PAGE and immunoblotting with the indicated antibodies. b Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either WT or ΔPEST Flag-SOCS3 and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. c Upper, Protein-equalised soluble cell extracts from HEK293 cells transfected with or without the indicated CIS and SOCS3 expression constructs and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel. Lower, Schematic of CIS and SOCS3. d Protein-equalised soluble cell extracts from HEK293 cells transfected with or without the indicated SOCS3 and Grap2 expression constructs and GFP-tagged cavin-1 as indicated were processed by IP with anti-Flag M2-agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the IP were also fractionated by SDS-PAGE for immunoblotting in parallel