Extended Data Figure 9. Cyclin D/CDK4-mediated phosphorylation of SPOP at the Ser6 residue promotes its binding with 14-3-3γ to reduce its poly-ubiquitination and subsequent degradation by APC/Cdh1.

a, A sequence comparison of conserved SP sites and putative 14-3-3γ binding motif in SPOP.

b, Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting.

c, d, In vitro kinase assays with recombinant Rb and SPOP as substrates and cyclin D1/CDK4, cyclin D2/CDK4 and cyclin D3/CDK4 as kinase complex were performed. BSA was used as a negative control where indicated.

e, IB analysis of WCL and immunoprecipitation (IP) derived from MDA-MB-231 cells transfected with indicated constructs, which were treated with/without palbociclib (1 μM) for 12 hours.

f, Streptavidin beads pull-down assay for biotin-labeled SPOP peptide with/without phosphorylation at the Ser6 residue to examine its in vitro association with 14-3-3γ.

g, IB analysis of WCL and GST pull-down precipitates derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting.

h, i, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) for 12 hours before harvesting.

j, k, IB analysis of WCL derived from 293T cells transfected with indicated constructs. 36 h post transfection, cells were treated with 20 μg/ml cycloheximide (CHX) as indicated time points (j). The protein abundance of SPOP-WT and S6A mutant were quantified by the ImageJ software and plotted accordingly (k).

l, p, IB of WCL and Ni-NTA pull-down products derived from the lysates of PC3 cells transfected with the indicated constructs. Cells were treated with MG132 (30 μM) for 6 hours before harvesting and lysed in the denaturing buffer for following assay.

m–o, IB analysis of WCL and IP derived from 293T cells transfected with indicated constructs and treated with MG132 (10 μM) and with/without palbociclib (1 μM) for 12 hours before harvesting.

q–s, IB of WCLs derived from PC3, BT549 and HeLa cells stably expressing sh14-3-3γ as well as shScr as a negative control.

t, IB of WCL derived from HeLa cells stably expressing shScr or sh14-3-3γ synchronized in M phase by nocodazole treatment prior to releasing back into the cell cycle for the indicated times.