Fig. 5: The ubiquitin-like domain of FRMD3 was required for the ubiquitination and degradation of vimentin through interaction with its head domain. | Cell Death & Disease

Fig. 5: The ubiquitin-like domain of FRMD3 was required for the ubiquitination and degradation of vimentin through interaction with its head domain.

From: FRMD3 inhibits the growth and metastasis of breast cancer through the ubiquitination-mediated degradation of vimentin and subsequent impairment of focal adhesion

Fig. 5

a RT-qPCR analysis of the mRNA expression levels of vimentin in the cells with FRMD3 overexpression or knockdown. b Western blot analysis of the stability of the vimentin protein in control and FRMD3-overexpressing MDA-MB-231 cells in the presence of cycloheximide (CHX, 50 μg ml−1) for the indicated times (left). The data were quantified using ImageJ software (right), and GAPDH was used for normalization. c Western blot analysis of vimentin protein levels in control and FRMD3-overexpressed cells under the treatment with the proteasome inhibitor MG132 (10 μM for 8 h), or not. d Co-IP analysis of the interaction between FRMD3 and vimentin in T47D cells and MDA-MB-231 cells with FRMD3 overexpression. e Co-IP analysis of interaction between the endogenous FRMD3 and vimentin in MCF10A cells. Isotype-matched IgG was used as a negative control. f Co-IP analysis of the interaction of endogenous FRMD3 with the indicated proteins in MCF10A cells. g Immunoprecipitated ectopically expressed GST-vimentin from MG132-treated MDA-MB-231 cells with FRMD3 overexpression were subjected to Western blot with anti-ubiquitin antibodies. h Ectopically expressed GST-vimentin in MG132-treated MDA-MB-231 cells with FRMD3 or FRMD3-Ubdel overexpression were immunoprecipitated with anti-GST, and then the immunoprecipitated was subjected to Western blot with the indicated antibodies. i Co-IP analysis of the interaction of FRMD3 with different regions of vimentin in MG132-treated MDA-MB-231 cells co-transfected with pcFlag-FRMD3 and full-length vimentin (FL) or individual truncated vimentin mutants (schematic diagram shown on the top). j IHC (left) or immunofluorescence staining (right) analysis of the levels of FRMD3 and vimentin in MDA-MB-231 cell xenograft in nude mice shown in Fig. 3e. Scale bar, 100 μm. N = 3 biologically independent replicates. The student’s t-test was used to estimate the significance of difference between two groups. Data were presented as means ± s.d ns not significant.

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