Skip to main content
. Author manuscript; available in PMC: 2022 Jul 6.
Published in final edited form as: Mol Cancer Res. 2021 Oct 5;20(1):62–76. doi: 10.1158/1541-7786.MCR-21-0108

Figure 2. GLI3 is targeted for proteasomal degradation by WT but not oncogenic mutant SPOP.

Figure 2.

a LNCaP whole cell lysates were subjected to immunoprecipitation (IP) with antibodies specific for GLI3 or isotype matched IgG. IPs were processed by western blot (WB) analysis using GLI3- and SPOP-specific antibodies as indicated. INPUT, 10% of lysate used in IP. b, c LNCaP cells were transfected with control (CNTL) or either of two different SPOP-specific shRNAs (SPOP KD1, SPOP KD2) (b) or increasing amounts of WT SPOP (c). Transfected whole cell lysates were processed by WB using antibodies specific for GLI3, SPOP, or TFIIEβ (loading control). In (c) cells were treated at 24 hrs post-transfection without or with MG132 (20 μM; 18 hrs) as indicated. d-e LNCaP cells transfected without or with Myc-tagged SPOP (M-SPOP; WT or mutant as indicated), were treated at 24 hrs post-transfection with MG132 (20 μM; 18 hrs). In (e), cells were also transfected with HA-tagged ubiquitin (Ub). Transfected whole cell lysates were subjected to IP with GLI3-specific antibodies and processed by WB using antibodies specific for GLI3, the Myc or HA epitopes, or TFIIEβ. INPUT, 10% of lysate used in IP. f LNCaP cells transfected without or with Myc-tagged SPOP (M-SPOP; WT or mutant as indicated) were treated at 24 hrs post-transfection without or with MG132 (20 μM;18 hrs). Transfected whole cell lysates were directly processed by WB using antibodies specific for GLI3, the Myc epitope, or TFIIEβ.