Fig. 2

Expression of RNF144B in ovarian cancer cell lines and preparation of a polyclonal antibody against human RNF144B. (a) RNF144B mRNA expression in cell lines and controls was evaluated using semi-quantitative PCR (qPCR). β-actin was used as a control. The expression is shown relative to normal ovarian tissue (b) Schematic diagram of RNF144B protein, including the RING finger, IBR: in between ring finger and TM: transmembrane domain. The peptide antibody was raised against the amino acid sequence from 224–243 (accession number: NP_877434.2) (c) HEK293T cells were transfected with RNF144B (pCDNA4 + MYC-RNF144B plasmid) and the lysates were immunoprecipitated with an anti-MYC antibody and blotted with RNF144B specific polyclonal antibody (1:5000); lane 1-Untransfected, lane 2-transfected (d) The lysate was also immunoprecipitated with RNF144B and blotted with an anti-MYC antibody (1:1000 dilution); lane 1-Antibody was incubated with a peptide corresponding to the epitope, lane 2- antibody alone (1:5000 dilution). (e) RNF144B protein expression levels in normal tissues (ovary, fallopian tube), immortalized cell lines (HOSE, FT33), and ovarian cancer cell lines (n = 10) were examined by Western blot. RNF144B protein band intensity was measured using ImageJ software and normalized to GAPDH. Western blot analysis was performed to confirm the stable transfection of the RNF144B gene in different clones of (f) PEO4 and (g) OVCAR3 cells. Empty vector pCDNA4/To/Myc/His-c was transfected into these cells as a control. Data values represent the relative RNF144B protein expression intensity normalized to that of GAPDH.