Figure 6.

AKNA is expressed after p53 restoration in HeLa and NCI-H1299 cells. (A) HeLa cells were transfected with a p53 expressing plasmid (pcDNA3-p53). After 24 h of transfection, cells were harvested, and total protein extracted using sample buffer. AKNA, CD40 and p53 protein levels were ascertained by western blot using AKNA anti-serum, anti-CD40 and anti-p53 antibodies. As expected p53 protein levels increased in cells transfected with the pcDNA3-p53 plasmid and AKNA levels become visible as well as CD40 protein levels in the same cells compared with the cells transfected with the control vector (pcDNA3). α-actinin was used as a loading control. (B) HeLa cells were transfected, fixed and stained for immunofluorescence analysis using AKNA anti-serum, anti-p53 and anti-CD40 followed by Alexa 488 and 555 secondary antibodies. p53 expressing cells showed a massive recovery in AKNA signal in the nucleus indicating the p53 dependent effect of AKNA. (C) HeLa cells were transfected with either pcDNA3 or the pcDNA3-akna plasmid, then cells were fixed and stained for immunofluorescence analysis using AKNA anti-serum and anti-CD40 antibodies followed by Alexa 488 and 555 secondary antibodies. Cells expressing AKNA exhibit a strong CD40 signal. (D) NCI-H1299 cells were transfected with pcDNA3-p53 plasmid. Twenty-four h after transfection, cells were collected and p53 expression assessed by western blot. (E) NCI-H1299 transfected cells were fixed and stained for immunofluorescence analysis using AKNA anti-serum and anti-p53 antibodies (Santa Cruz Biotechnology) followed by Alexa 488 and 555 secondary antibodies. p53 expressing cells show an evident AKNA signal. Slides were mounted using Prolong DAPI media (Molecular probes) and observed in a confocal NIKON Eclipse Ti microscope. All images were acquired under the 40× objective using a NIKON camera.