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. 2022 Mar 7;12:3687. doi: 10.1038/s41598-022-07642-1

Figure 3.

Figure 3

UCHL5 stabilizes Axin1 in a non-enzymatic fashion. (a) Western blot analysis using HeLa cells. Cells were transfected with HA-Axin1 (0.5 μg) and Flag-UCHL5 plasmids (2 μg and 3 μg). Cell lysates were then immunoblotted to detect the level of HA-Axin1, Flag-UCHL5, and GAPDH proteins. (b) Western blot analysis using HeLa cells. Flag-UCHL5 plasmids (2 μg and 3 μg) were introduced into cells. Cell lysates were then subjected to immunoblotting to detect endogenous Axin1, Flag-UCHL5, and GAPDH proteins. (c) Western blot analysis using Co, KD1, and KD2 HeLa cells. Endogenous Axin1, Actin, and UCHL5 levels were determined by anti-Axin1, anti-Actin, and anti-UCHL5. (d) Pulse-chase test using HeLa cells. The level of endogenous Axin1 protein was measured over time under the translation-blocked condition by cycloheximide (100 μg/ml). The band intensity of Axin1 was measured with ImageJ software and was normalized to Actin. (e) qPCR analysis for the expression of Axin1 mRNA in Co, KD1, and KD2 HeLa cells. The level of Axin1 mRNA was normalized by GAPDH mRNA. (f) Western blot analysis using Co and KD1 HeLa cells. Cells were transfected with myc-Axin1 (1 μg), and treated with either NaCl or LiCl (50 mM). Cell lysates were then subjected to immunoblotting to detect myc-Axin1, active ß-catenin, UCHL5, and GAPDH proteins. (g) Western blot analysis using HeLa cells. The requirement of deubiquitinating activity for the Axin1 stabilization was determined by comparing WT and Mut.UCHL5. HA-Axin1 (0.5 μg), WT UCHL5 (1 μg and 2 μg), and Mut. UCHL5 (1 μg and 2 μg) were introduced into KD1 HeLa cells. Cell lysates were immunoblotted with antibodies against HA, Actin, and UCHL5. The band intensity of HA-Axin1 was measured with ImageJ software and was normalized to GAPDH. (h) TOPflash assay using HeLa cells. Cells were transfected with TOPflash reporter (250 ng) and TK-Renilla reporter (50 ng), together with either WT UCHL5 or Mut.UCHL5. 8 h after transfection, cell culture media were replaced with Wnt3a CM and cells were further incubated for 16 h. (i) Western blot analysis using HeLa cells. KD1 HeLa cells were treated with either MG132 (20 μM) and Chloroquine (100 μM) for 5 h. Then, cell lysates were immunoblotted with antibodies for Axin1, UCHL5, and Actin. (j) In vivo ubiquitination assay using HeLa cells. myc-Axin1 (2 μg) was transfected alone or with Ub-HA (2 μg), together with either Flag-UCHL5 (2 μg and 4 μg) or Flag-Mut. UCHL5 (2 μg and 4 μg). After that, Cells were treated with MG132 (20 μM) for 5 h and then lysed and subjected to immunoprecipitation with anti-myc. (k) In vivo ubiquitination assay using HeLa cells. myc-Axin1 (2 μg) was transfected into WT and two KD HeLa cells (KD1 and KD2), alone or with Ub-HA (2 μg). Transfected cells were treated with MG132 (20 μM) for 5 h and then subjected to immunoprecipitation with anti-myc. The data from qPCR analysis (e) and TOPflash (h) and displayed as means ± SD and show a representative of multiple independent experiments (n = 3 biological independent experiments). ns, not significant and ** P < 0.005. from two-tailed unpaired t-test (e, h).