Fig. 7

PDE5A-500aa promotes PIK3IP1 de-ubiquitination activity. A WB analysis of PDE5A-500aa and PIK3IP1 protein level in KYSE30 cells treated with vector and incremental circPDE5A-flag plasmid. PIK3IP1 mRNA level was assessed by PCR and nucleic acid electrophoresis. B WB analysis of PDE5A-500aa and PIK3IP1 protein level in ECA109 cells treated with sh-NC, sh-circPDE5A-1, and sh-circPDE5A-2 plasmids. PIK3IP1 mRNA level was assessed by PCR and nucleic acid electrophoresis. C Protein biosynthesis inhibition in KYSE30 or ECA109 cells with 20 μg/mL cycloheximide (CHX). WB analysis of PIK3IP1 protein levels in ESCC cells overexpressing or knocking down circPDE5A at various time points. D Inhibition of protein degradation with MG-132 (40 μM) for 8 h. WB analysis of PIK3IP1 protein levels in circPDE5A knockdown ECA109 cells. E ESCC cells co-transfected with HA-Ub and Myc-PIK3IP1, treated with MG132. Ubiquitination levels of PIK3IP1 protein detected by IP and WB after treatment with vector, circPDE5A-flag, circPDE5A-Mut, and PDE5A-500aa-flag plasmids. F ESCC cells transfected with HA-Ub and Myc-PIK3IP1, treated with MG132. Ubiquitination level of PIK3IP1 protein detected by IP and WB after treatment with sh-NC, sh-PIK3IP1-1, and sh-PIK3IP1-2 plasmids. G IP and WB detection of the ubiquitination level of the PIK3IP1 protein in circPDE5A overexpressing ESCC cells co-transfected with HA-tagged different ubiquitin chain vectors with Myc-PIK3IP1 and treated with MG132. H ESCC cells co-transfected with HA-K48-Ub and circPDE5A, treated with MG132. Ubiquitination level of PIK3IP1 protein detected by IP and WB after treatment with different Myc-tagged PIK3IP1 lysine site-indicated mutants. I ESCC cells transfected with HA-K48-Ub and treated with MG132. Ubiquitination level of PIK3IP1 protein detected by IP and WB after treatment with vector, circPDE5A, and circPDE5A + Myc-PIK3IP1 (K198R) plasmid. J Detection of PDE5A-500aa and PIK3IP1 protein level in ESCC cells treated with circPDE5A, PIK3IP1, or PIK3IP1 (K198R) mutant by WB. K Detection of PDE5A-500aa and PIK3IP1 protein level in ESCC cells treated with sh-circPDE5A, PIK3IP1, or PIK3IP1 (K198R) mutant by WB. ***P < 0.005