Fig. 5: Glutamine deprivation inhibits SPOP dimerization and promotes SPOP self-ubiquitylation.

a, b MDA-MB-231 cellswere cultured with indicated percentage of glutamine for 24 h (a) or glutamine-free medium at different time points (b), for immunoblotting. c MDA-MB-231 cells were cultured in glutamine-free medium for 6 h with MG132 or Chloroquine (CQ) for another 6 h or 24 h, followed by immunoblotting. d MDA-MB-231 cells were transfected with FLAG-SPOP plasmid, cultured in glutamine-free medium with MG132, then for immunoblotting. e, f MDA-MB-231 cells were cultured in glutamine-containing or glutamine-free medium with addition of CHX, harvested at indicated time points for immunoblotting. (e). The band density of SPOP was quantified and normalized to α-tubulin (mean ± SD, n = 3) (f). g HEK293 cells were transfected with indicated plasmids, cultured in glutamine-containing or glutamine-free media for 12 h with or without MG132. Cells were lysed under denaturing conditions, followed by Ni-beads pulldown, and immunoblotting with anti-FLAG antibody. h HEK293 cells were transfected with indicated plasmids, cultured in glutamine-containing or glutamine-free media, followed by immunoprecipitattion with FLAG-agarose beads and analyzed by immunoblotting. i HEK293 cells were transfected with indicated plasmids, cultured in glutamine-containing or glutamine-free media, and lysed under denaturing conditions, followed by Ni-beads pulldown, immunoblotting for ASCT2. j Endogenous SPOP protein in SK-BR-3 cell lysates were pulled down with anti-SPOP antibody, and associated GRK2 were detected by immunoblotting with anti-GRK2 Ab (top, IP). Cell lysates were subjected to immunoblotting (bottom, input). k MDA-MB-231 cells were cultured in glutamine-containing or glutamine-free media in the presence or absence of GRK2 inhibitor PX (10 μM) for 6 h, and analyzed by immunoblotting. l MDA-MB-231 cells were transfected with siRNAs targeting GRK2 or scramble control siRNA, then cultured in glutamine-containing or glutamine-free media for 6 h, and analyzed by immunoblotting. m MDA-MB-231 cells were cultured in glutamine-free medium for indicated time points and analyzed by immunoblotting. n MDA-MB-231 cells were transfected with indicated plasmids, then cultured in glutamine-containing or glutamine-free media for indicated time points, followed by immunoblotting. o–q HEK293 cells were first transfected with siRNA targeting SPOP (3′-UTR) (o, p) then with indicated plasmids (o–q). Cells were cultured with glutamine-free medium for 12 h (o, p) or, cultured in glutamine-containing or glutamine-free media in the absence or presence of PX for 12 h (q), followed by immunoprecipitation with FLAG-agarose beads and analyzed by immunoblotting (o) or lysed under denaturing conditions, followed by Ni-beads pulldown, and analyzed by immunoblotting for FLAG antibody (p) or for ASCT2 (q). Source data are provided as a Source Data file.