Fig. 7

AFAP1-AS1 interacted with AUF1, thereby activated the translation of ERBB2. a Nuclear fraction experiment and qRT-PCR experiments were performed to detect the relative distribution of AFAP1-AS1 in nuclear and cytoplasm of SKBR-3-TR and BT474-TR cells. b Prediction of 911-1190 nt AFAP1-AS1 structure was based on minimum free energy (MFE) and partition function (http://rna.tbi.univie.ac.at/). c Immunofluorescence was performed to identify the subcellular distribution of AUF1 protein in SKBR-3-TR and BT474-TR cells. d RNA pulldown followed by western blotting was done with AFAP1-AS1 probe to verify the direct interaction between AUF1 and AFAP1-AS1. GAPDH served as internal control. e RIP was performed using anti-AUF1 and control IgG antibodies, followed by qRT-PCR to examine the enrichment of DANCR and U6. U6 served as negative control, ^***P < 0.001. f qPCR showed that knockdown of AFAP1-AS1 showed no influence on AUF1 mRNA level. g Knockdown of AUF1 decreased HER-2 protein expression level without influencing the ERBB2 mRNA level. h Western blotting revealed that knockdown of AUF1 abrogated the increased HER-2 expression induced by overexpression of AFAP1-AS1. i-j The endogenous AUF1 binding to ERBB2 mRNA was increased by overexpression of AFAP1-AS1in SKBR-3 cells(i), while decreased by knockdown of AFAP1-AS1 in SKBR-3-TR and BT474-TR cells(j), ^*P < 0.05, ^**P < 0.01. k SKBR-3-TR and BT474-TR cells silenced with AFAP1-AS1 were cultured with 20 μg/ml Cycloheximide (CHX) for 0–60 min, then subjected to western blotting analysis