Fig. 8

PDE5A-500aa stabilizes PIK3IP1 via USP14-mediated de-ubiquitination. A Investigation of PIK3IP1 interacting proteins and de-ubiquitinating enzymes. Left panel: Immunoprecipitation (IP) of whole cell lysates from PIK3IP1 overexpressing 293 T cells with anti-PIK3IP1 antibody, followed by visualization of the pull-down proteins by WB and silver staining. Right panel: Venn diagram illustrating de-ubiquitinating enzymes potentially bound to PIK3IP1 identified by LC–MS/MS. B Specific information on the five de-ubiquitinating enzymes identified by LC–MS/MS, ranked in order of enriched abundance. C Secondary mass spectra of the USP14 protein identified by LC–MS/MS. D Validation of the interaction between PIK3IP1 and USP14 in ESCC cells by Co-IP. E Visualization of docking patterns and key residue interfaces for protein–protein interaction between PIK3IP1 and USP14, obtained using PyMOL software. F Immunofluorescence analysis showing co-localization of PIK3IP1 and USP14 proteins in the cytoplasm of ESCC cells. Bar represents 10 μm. G IP and WB to detect the interaction between PIK3IP1 and USP14 in circPDE5A overexpressing and knockdown ESCC cells. H ESCC cells transfected with HA-K48-Ub and Myc-PIK3IP1, treated with MG132. Detection of PIK3IP1 ubiquitination levels in USP14 overexpressing or knockdown ESCC cells using IP and WB. I ESCC cells transfected with HA-K48-Ub and Myc-PIK3IP1, treated with MG132. Detection of PIK3IP1 ubiquitination level after treatment with the flag-tagged USP14 indicated mutant (flag-USP14-C114A) using IP and WB. J ESCC cells transfected with HA-K48-Ub and Myc-PIK3IP1, treated with MG132. Detection of PIK3IP1 ubiquitination level after treatment with circPDE5A or sh-USP14 plasmid using IP and WB. K ESCC cells transfected with HA-K48-Ub and Myc-PIK3IP1, treated with MG132. Detection of PIK3IP1 ubiquitination level after treatment with sh-circPDE5A or USP14 plasmid using IP and WB. L WB analysis of USP14 and PIK3IP1 level in ESCC cells after treatment with circPDE5A or sh-USP14 plasmid. M WB analysis of USP14 and PIK3IP1 level in ESCC cells after treatment with sh-circPDE5A or USP14 plasmid