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Fig. 8 | Molecular Cancer

Fig. 8

From: CircSTX6 promotes pancreatic ductal adenocarcinoma progression by sponging miR-449b-5p and interacting with CUL2

Fig. 8

CircSTX6 physically interacts with CUL2 and participates in the regulation of HIF1A. a CircSTX6 and oligo probes were incubated with proteins extracted from PDAC cells for RNA pull-down assays. Proteins pulled down were used for silver staining, and a specific band appeared between 75 and 100 kDa (arrow). b Levels of CUL2 protein were detected in the proteins pulled down by circSTX6 and oligo probes. c RIP assays were performed using antibodies against CUL2 and IgG. d-f Levels of CUL2 mRNA and protein in circSTX6-overexpressing and circSTX6-knockdown PDAC cells were quantified by qRT–PCR and western blotting. β-actin served as the internal control. g The levels of interacting proteins immunoprecipitated by CUL2 were detected in circSTX6-overexpressing PDAC cells. h CUL2 was truncated (1–186 aa, 187–372 aa, 373–568 aa, and 569–744 aa) to identify the specific fragment of CUL2 that bound to circSTX6. RIP assays were performed to detect the enrichment of circSTX6 in cells transfected with full-length and truncated flag-tagged constructs. i HIF1A was first immunoprecipitated, and the ubiquitination level of HIF1A was detected in PDAC cells transfected with the circSTX6 plasmid. j The expression level of HIF1A was detected in circSTX6-overexpressing or circSTX6-knockdown PDAC cells. k Representative HIF1A IHC images of a tissue microarray containing 97 pairs of PDAC and corresponding noncancerous pancreatic tissues. Original magnification 75× and 300×. Scale bar = 100 μm. l The protein level of HIF1A was significantly increased in PDAC tissues. (Values are expressed as the means ± SDs; *P < 0.05, **P < 0.01 and ***P < 0.001)

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