Figure 2

PLD2 antisense oligonucleotides reduce endogenous PLD2 mRNA expression and protein levels in SN56 cells. SN56 cells were transfected with either control or antisense oligonucleotides as described in the Experimental Procedures. Two days later PLD2 gene expression was assessed by Northern blot analysis (A-C) as described in Fig. 1. G3PDH was used for normalization (A-C). For Western blots 40 μg of lysate protein was subjected to SDS-PAGE. A polyclonal anti-human PLD2 antibody was used first to detect PLD2, the blot was stripped and rehybridized with a monoclonal anti-mouse actin antibody for normalization (D,E). A. Concentration-response curve to the antisense oligonucleotide. B. Comparison of the effects of the antisense and control oligonucleotides on PLD2 expression. The cells were transfected with 200 nM of either control or antisense oligomer as indicated. C. Quantification of results presented in B. Intensity of the hybridization bands was normalized to that of G3PDH. The antisense oligomer reduced PLD2 expression by 34%, ^*p < 0.05. Data shown are from 3–6 experiments and expressed as average ± SEM. D. The antisense oligomer reduced PLD2 protein expression as compared to mock-treated and control oligomer-transfected cells. E. The intensity of the PLD2 bands was quantified using NIH Image 1.62 and normalized to that of actin. The antisense oligomer reduced the PLD2 protein level by 28% compared to controls, ^*p < 0.05. The results are from 3 experiments and expressed as average ± SEM.