Figure 3: SOCS2 interacts with NDR1 and promotes its ubiquitin-mediated degradation.

(A,B) FLAG-SOCS2-ΔSB and Myc-NDR1 were expressed in MEFs either alone or together. Lysates were immunoprecipitated with anti-Myc (9E10) antibody (A) or anti-FLAG M2 antibody (B) and subjected to SDS–PAGE followed by IB. (C) HEK293T cells grown to ~80% confluency were harvested. Lysates (2 mg) were subjected to IP with anti-IgG or anti-SOCS2 antibody followed by IB with the indicated antibodies. Values on the right are densitometric quantifications with the corresponding “Input” band taken as 1. (D) MEFs were transfected with FLAG-SOCS2 plasmid as shown. Cells were treated with 10 μM MG132 or an equal volume of DMSO for 16 hrs before harvesting. Lysates were prepared and IB was done as previously. (E,F) Plasmids for HA-Strep-Ubiquitin and Myc-NDR1 were co-expressed in MEF cells with FLAG-SOCS2 (E) or FLAG-SOCS2 and FLAG-SOCS2-ΔSB (F) as shown. Cells were treated with 10 μM MG132 or an equal volume of DMSO for 16 hrs before harvesting. Lysates were prepared from 36 hrs post-transfected cells and subjected to pull-down using StrepTactin resin followed by IB. (G) Plasmids for HA-Strep-Ubiquitin-K48 or K48R and Myc-NDR1 were co-expressed in HEK293T cells with FLAG-SOCS2 as shown. Cells were treated with 10 μM MG132 or an equal volume of DMSO for 16 hrs before harvesting. Lysates were prepared from 36 hrs post-transfected cells and subjected to pull-down using anti-Myc (9E10) antibody followed by IB. Lysates were probed by IB. Full-length blot of 3D (NDR1) are presented in Supplementary Figure S4.