Figure 2.

Jade-1 reduces β-catenin protein abundance. (a) Differential regulation of β-catenin constructs by Jade-1. Myc-tagged β-catenin and Flag-tagged Jade-1 (J1) constructs were transiently transfected into 293T cells. β-catenin construct schematics are shown in Supplementary Information, Fig. S1a. WCL were probed for β-catenin and α-tubulin protein. Expression of Jade-1 was detected by immunoblotting using Flag antibody. Densitometry was performed with NIH ImageJ using α-tubulin as loading control. Representative immunoblot of 5 experiments. (b) Jade-1 regulates the cytosolic and nuclear fractions but not the membrane fraction of endogenous β-catenin. The 293 uninfected parental cells (P) or cells infected with empty vector (Vec) or Jade-1 shRNA lentivirus (Jsh) were subjected to cell fractionation. Cytosolic, nuclear and membrane fractions were probed for β-catenin. α-tubulin, fibrillarin and E-cadherin served as loading controls and markers of cytosolic, nuclear and membrane fractions, respectively. These control blots were generated by cutting the membrane at the expected molecular weights of the controls and immunoblotting separately. One of 2 similar experiments is shown. (c) Jade-1 reduces cytosolic and nuclear pools of endogenous β-catenin. Extracts of clonal 786-O renal cancer cell lines stably expressing empty vector (Vec) or Jade-1 (J9 and J18)⁴,⁵ were probed for β-catenin. α-tubulin, fibrillarin and N-cadherin served as markers of cytosolic, nuclear and membrane fractions and as loading controls, respectively. These control blots were generated by cutting the membrane at the expected molecular weights of the controls and immunoblotting separately. Jade-1 was detected using polyclonal Jade-1 antibody. Representative immunoblot of 3 experiments. The effect of stably-transfected Jade-1 on β-catenin abundance appears to be dose dependent. (d) Jade-1 silencing substantially prolongs the half-life of endogenous cytosolic β-catenin. Digitonin was used to extract the cytosolic pool of β-catenin. Digitonin is a gently acting detergent that at low concentrations selectively punctures 8-10 nm holes in the sterol rich plasma membrane, allowing leakage of cytosolic proteins of up to 200 kDa mass¹⁰ (Upper panels), Digitonin extracts of 293 cells infected with empty vector or JshRNA lentiviral vector and pretreated with 20 μM emetine were probed for β-catenin by immunoblotting. (Lower panel), Percent β-catenin remaining in the digitonin-extracted fraction was analyzed by densitometry after normalizing to α-tubulin. Graph shows mean result of 4 experiments. Error bars = SEM. (e) GSK-3β silencing mitigates Jade-1 regulation of endogenous β-catenin. The 293T cells were transiently cotransfected with Flag-tagged Jade-1 (J1) and GSK-3β shRNA plasmid (GSK-3βsh) or empty vector (Vec) and GFP. One day post transfection, cells were sorted using fluorescence-activated cell sorting (FACS) and were replated for 48 hrs. Protein extracts from cells were probed for β-catenin using monoclonal β-catenin antibody. GSK-3β and Jade-1 proteins were examined by immunoblotting with polyclonal GSK-3β and Jade-1 antibodies, respectively. α-tubulin, fibrillarin and E-cadherin served as loading controls and markers of cytosolic, nuclear and membrane fractions, respectively. One of 2 similar experiments is shown. (f) Chemical inhibition of GSK-3β mitigates Jade-1 regulation of endogenous β-catenin. Transiently transfected 293T cells were serum starved for 12-16 h and treated with lithium chloride (10 mM) or BIO (100 nM) for an additional 12 h. β-catenin was detected using β-catenin antibody. Representative immunoblot of 4 experiments. (g) Jade-1 regulation of endogenous β-catenin in Wnt-on and Wnt-off status. HeLa parental cells (P) or cells infected with empty vector (Vec) or Jade-1 shRNA (Jsh) lentivirus were treated for 4 h with Wnt-3a (100 ng) with or without DKK1 (100 ng). The cytosolic fractions were probed for β-catenin and α-tubulin protein. Representative immunoblot of 4 experiments.