Fig. 4.

Cavin-1–SOCS3 interaction occurs independently of the PTyr binding capacity of the SH2 domain. a HEK293 cells transfected with expression constructs encoding Flag-SOCS3 and GFP-tagged cavin-1 as indicated were treated with or without Tyr phosphatase inhibitors sodium orthovanadate (Van: 1 mM) for 1.5 h and then hydrogen peroxide (H₂O₂: 0.2 mM) for an additional 30 min prior to harvesting. Protein-equalised soluble cell extracts were then 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. b Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding either WT or R71K-mutated 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 Protein-equalised soluble cell extracts from HEK293 cells transfected with expression constructs encoding WT Flag-SOCS3 and GFP-tagged cavin-1 as indicated 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 or a scrambled control (Scr) and streptavidin–agarose beads prior to SDS-PAGE and immunoblotting with the indicated antibodies. Whole-cell lysates from the samples used in the pull-down were also fractionated by SDS-PAGE for immunoblotting in parallel