Fig. 5: Moesin and SKP2 interact with and positively regulate each other’s expression at the posttranscriptional level.

A Immunoblot showing the expression levels of SKP2, His-Moesin, and β-actin in MCF7 cells expressing either vector or different doses of His-Moesin. B Immunoblot showing the expression levels of Moesin, SKP2, and tubulin in MCF7 cells expressing either scramble shRNA (NS) or Moesin specific two independent shRNAs. C Real-time qRT-PCR showing relative mRNA levels of SKP2 in MCF7 cells expressing either scramble shRNA (NS) or two independent Moesin specific shRNAs. β-actin was used as internal control. D Control (NS) and Moesin-depleted MCF7 cells were grown in the presence or absence of 5 µM MG132 for 6 h as indicated. Whole-cell protein extracts were immunoblotted for the indicated proteins. E Control (NS) and Moesin-depleted MCF7 cells were grown in the presence of 5 µM MG132 for 6 h before harvesting. Whole-cell protein extracts were immunoprecipitated with either anti-IgG (control) or anti-SKP2 antibody. Immunoprecipitates were immunoblotted for K48-linked ubiquitin and input protein extracts were immunoblotted for the indicated proteins. F MCF7 cells were transfected with either vector or His-Moesin for 36 h. Transfected cells were then treated with 5 µM MG132 for 6 h. Whole-cell protein extracts were immunoprecipitated with anti-IgG or anti-FBXW2 antibody. Immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins. G Immunoblot showing the expression levels of Moesin, FLAG-SKP2, and Tubulin in MCF7 cells expressing either vector or different doses of FLAG-SKP2. H Real-time qRT-PCR showing relative mRNA levels of Moesin in MCF7 cells expressing either vector or FLAG-SKP2. β-actin was used as internal control. I MCF7 cells were transfected with indicated plasmids for 36 h. Transfected cells were then treated with 5 µM MG132 for 6 h. His-Moesin were pulled down by using Ni-NTA beads from whole-cell protein extracts. Pulled down proteins and input protein extracts were immunoblotted for the indicated proteins. J MCF7 cells were harvested and whole-cell lysates were immunoprecipitated with either anti-IgG or anti-SKP2 antibody. Then, immunoprecipitates and input protein extracts were immunoblotted for the indicated proteins. K Recombinant purified GST and GST-Moesin proteins were incubated with Ni-NTA bead-bound FLAG-SKP2 at indicated combinations for 2 h at room temperature. Then, Ni-NTA beads were washed and pulled down proteins were eluted with SDS buffer. Elutes and input proteins were immunoblotted for the indicated proteins. L MCF7 cells were transfected with indicated plasmids for 36 h followed by addition of 5 µM MG132 for 6 h and lysed under denaturing condition. Ubiquitinated proteins were pulled down with Ni-NTA beads from whole-cell protein extracts. Pulled down proteins and input protein extracts were immunoblotted for the indicated proteins. M In vitro ubiquitination experiment was performed by using recombinant purified proteins. All the purified components and ATP were incubated with ubiquitination buffer (50 mM Tris, pH 8.0, 5 mM MgCl₂, 1 mM β-mercaptoethanol, and 0.1% Tween 20) in indicated combination for 2 h at room temperature. Then, SDS sample buffer was added to stop the reaction and boiled for 5 min. The samples were then resolved in SDS-PAGE followed by western blotting with anti-GST antibody. N Trypan blue exclusion assay in MCF7 cells expressing either NS or shRNAs against SKP2 or FBXW2 or both. O Long-term colony formation assay in MCF7 cells expressing either NS or shRNAs against SKP2 or FBXW2 or both. P Quantification of colonies from (O). Q Invasion assay in MCF7 cells expressing either NS or shRNAs against SKP2 or FBXW2 or both. Scale bars = 100 µM. R Quantification of invaded cells from (Q). S Scratch wound healing assay in MCF7 cells expressing either NS or shRNAs against SKP2 or FBXW2 or both. Scale bars = 100 µM. T Quantification of migrated cells from (S).