Abstract 2209: Characterization of FABP5 antibodies in prostate

FABP5, a member of a family of small proteins (~15 kDa) that transport lipids, is emerging as an important biomarker because it is differentially expressed in prostate cancers (PrCa) compared to uninvolved prostate glands and lipids are important to the progression of prostate cancer. This study was to select an antibody (Ab) for use in immunostaining (IHC) FABP5 in formalin fixed paraffin embedded (FFPE) tissue and also an Ab for use in Western blotting (WB). A mouse monoclonal antibody (mAb) clone A9 to FABP5 from Santa Cruz (SC) Biotechnology, Inc. and a rabbit polyclonal antibody (pAb) to FABP5 from Abcam (AB) (cat. # ab37267) were tested. Six prostate cell lines (PC3, DU145, 22Rv1, MDA PCa 2b, LNCaP, and normal primary prostate epithelial cells (NH Pri Pro) were used to study protein and mRNA levels of FABP5. Protein expression of FABP5 was determined by WB of whole cell lysates as well as cytoplasmic and nuclear lysates. GAPDH, a “housekeeping” gene expressed at similar levels in most types of cells, was used as a loading control for WB. Cells from the six prostate cells lines also were embedded in HistoGel, processed to FFPE and immunostained. The prostate cells were transfected with FABP5 siRNA for WB with aliquots of the transfected prostate cells processed to FFPE. qrtPCR was performed to measure mRNA levels. The expression of whole cell lysates on WB probed with the AB pAb to FABP5 was NH Pri Pro > PC3 > DU145 > 22Rv1 >> MDA PCa 2b > LNCaP compared to NH-Pri Pro > PC3 > DU145 > 22Rv1 when probed with the SC mAb. WB of the cytoplasmic fractions from each cell line has a similar pattern. In HistoGel sections immunostained with AB pAb, the FABP5 cytoplasmic expression was PC3 > DU145 > 22Rv1 > MDA PCa 2b > NH Pri Pro >> LNCaP. These levels of expression among the cell lines followed a similar trend in evaluation of immunostaining of cell membranes, nuclear, and the perinuclear area. In HistoGel sections immunostained with SC mAb, the FABP5 expression was PC3 > DU145 with weak signals in 22Rv1 > MDA PCa 2b > NH Pri Pro >> LNCaP. A recombinant blocking peptide (RBP) to FABP5 strongly inhibited the immunostaining with the antibodies to FABP5 in FFPE sections from archival radical prostatectomy specimens. This RBP is the same sequence as the peptide used to produce the AB antibody. No RBP to FABP5 was available from SC. Incubation with the RBP strongly inhibited the extent of immunostaining with FABP5 SC mAb. Similarly, immunostaining of the 6 cell lines with the AB pAb following incubation with RBP strongly inhibited staining. In analysis of mRNA levels, the expression of FABP5 was PC3 > 22Rv1 > > DU145 > MDA PCa 2b >> LNCaP. We demonstrated by siRNA and the use of a RBP that both these antibodies are specific and sensitive for detecting FABP5 by immunostaining but the AB Ab is more useful for WB. These results are presented with the IHC of FABP5 in normal and uninvolved prostate glands in PrCa. Citation Format: Dennis A. Otali, Denise K. Oelschlager, James Kearns, Sandra M. Gaston, William E. Grizzle. Characterization of FABP5 antibodies in prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2017-2209