Fig. 2: The lack of fortilin destabilizes CTNNA3 and causes it to be degraded via a proteasome-mediated pathway.

THP1^WT-fortilin THP1 cells expressing wild-type fortilin, THP1^KO-fortilin cells in which the fortilin genes have been deleted using Crispr-Cas9 technology, GFP^strep-tag strep-tagged recombinant green fluorescence protein, FT^strep-tag strep-tagged recombinant human fortilin protein, * degradation products, IB immunoblot, TCE 2,2,2-trichloroethanol, DAPI 4’,6-diamidino-2-phenylindole, siRNA^fortilin small interfering RNA against fortilin, siRNA^Control the non-targeting pool of siRNAs, A.U. arbitrary unit, IB immunoblot, α-CTNNA3 anti-CTNNA3 Ab, MG132 carbobenzoxy-L-leucyl-L-leucyl-L-leucine, a proteasome inhibitor. a Characterization by western blot analysis of THP1^WT-fortilin and THP1^KO-fortilin cells. Total proteins were visualized in the gel using TCE. b THP1^WT-fortilin and THP1^KO-foritlin cells were seeded on a chamber slide and subjected to immunocytochemistry using α-CTNNA3 and α-fortilin Abs. The nucleus was counter-stained by DAPI. The intensities of CTNNA3 and fortilin signals were evaluated by confocal microscopy using the same imaging conditions. Long scale bar = 100 µm. Short scale bar = 10 μm. c Silencing of fortilin by siRNA^fortilin. Fortilin mRNA expression levels was evaluated by RT-qPCR normalized to that of 18S rRNA after 293T cells were transfected with the siRNAs. Data are expressed as means ± s.d. (n = 3 biological replicates), and they were analyzed by two-tailed, unpaired Student’s t test. d Western blotting analysis of fortilin protein expression levels after 293T cells were transfected with the siRNAs. Total proteins were visualized by TCE staining. e Densitometric quantification of fortilin protein signals using ImageJ software. The fortilin protein expression indices (in A.U.) were determined by dividing the signal intensity of the immunoblotted fortilin band by that of the total protein bands identified by TCE staining. f Time-course analysis using western blot analysis of CTNNA3 expression levels in 293T cells. g Densitometric quantification of CTNNA3 signals using ImageJ software. The CTNNA3 expression indices were calculated by dividing the signal intensities of the immunoblotted CTNNA3 bands by those of the total protein bands identified by TCE staining. They were expressed in A.U. after normalizing the indices to time 0. The percentage reduction of CTNNA3 proteins at 36 h is shown in the graph. Data are expressed as means ±s.d. (n = 3 biological replicates), and they were analyzed by two-tailed, unpaired Student’s t tests.