Fig. 4: PPIL2 modulated SNAI1 stability and ubiquitination

a Western blot assay showed that the decreased expression of SNAI1 in PPIL2-transfected ZR-75-30 cells was reversed 4 h after treatment with 10 μM MG132. b Western blot assay that showed overexpression of PPIL2 resulted in a shorter half-life of SNAI1 in ZR-75-30 cells treated with CHX. c Co-IP assay showed that the ubiquitin levels associated with SNAI1 were reduced when PPIL2 was silenced in MCF-7 cells. d Immunoblot analysis of lysates in 293T cells transfected for 24 h with GFP-SNAI1, along with HA-K63-Ub, HA-K48-Ub, and Flag-PPIL2, followed by immunoprecipitation with anti-GFP magnetic beads. e The cells were collected 24 h after transfection of Flag-PPIL2 and GFP-SNAI1, and the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Themofisher, Beijing, China) were used to extract nuclear protein and cytoplasmic protein. Western blot assay showed that elevated PPIL2 reduced nucleus ___location of SNAI1 in ZR-75-30 cells. f Cytoplasmic protein and nuclear protein were extracted for separate immunoprecipitation. The results showed that PPIL2 increased the ubiquitination of SNAI in the nucleus but had no effect on the ubiquitination of SNAI1 in the cytoplasm. Western blot assay showed that elevated PPIL2 increased the enrichment of SNAI1 ubiquitination in the nuclear but not the cytoplasmic fractions. g Co-IP assay showed that PPIL2 prompted the ubiquintination of SNAI1 in the nucleus in ZR-75-30 cells after incubation with 5 ng/ml Leptomycin B for 3 h