Fig. 5

The mechanism of circPLCE1-411 inhibits NF-κB signaling. A Western blot analysis of immunoprecipitation using Flag, HA or RPS3 antibodies in HCT8 and DLD1 cells transfected with circPLCE1-flag vector or HA-HSP90α vector with indicated antibodies. B Western blot analysis of HCT8 and DLD1 cells transfected with circPLCE1-ATGmut or increasing circPLCE1 vector with indicated antibodies. Below, RT-PCR analysis of RPS3 mRNA; 18S rRNA serves as a loading control. C Western blot analysis of RPS3 protein levels in HCT8 cells transfected with circPLCE1-ATGmut or circPLCE1 with cycloheximide (CHX, 50 μg/ml) treatment at the indicated times. D Western blot analysis of RPS3 protein levels in HCT8 cells transfected with circPLCE1-ATGmut or circPLCE1 with MG132 (25 μM) for 12 h. E Western blot analysis of RPS3 ubiqitin levels after immunoprecipitation using RPS3 antibodies in HCT8 cells transfected with circPLCE1-ATGmut, circPLCE1, shNC or shcircPLCE1 vector. MG132 (25 μM) was added 6 h before harvest. F HEK293T cells were transfected with the indicated plasmids and MG132 (25 μM) was added simultaneously during transfection. Cell lysates were immunoprecipitated with anti-His antibody and then immunoblotted by the indicated antibodies. G Western blot analysis of immunoprecipitation using HA antibodies in HEK293T cells transfected with HA-HSP90α (FL), HA-HSP90α (N) and HA-HSP90α (C) with indicated antibodies. H Western blot analysis of p-P65 and P65 protein levels from whole-cell, nuclear, and cytoplasmic extracts in HCT8 cells transfected with the indicated plasmids. GAPDH, β-actin and Lamin A served as loading control. I Transcription factor binding assay of P65 in nuclear extracts obtained from HCT8 cells transfected with the indicated plasmids, n = 3. Values are represented as mean ± SD. ***p < 0.001, by 2-tailed Student’s t test (I)