Img 5143a

Analysis of viral protein expression was done essentially as described previously (Peters et al., 2005 (link)). In brief, cell lysates were prepared using lysis buffer (RIPA plus protease inhibitors). Viral proteins were probed with anti-Gag (Heinkelein et al., 2002 (link)), anti-Pol and anti-Env (Imrich et al., 2000 (link)) mouse monoclonal antibodies after separation in sodium dodecyl sulfate-containing 8% polycrylamide gels and semi-dry blotting onto Hybond ECL membranes (Pharmacia). Protein bands were detected using horseradish-coupled secondary antibodies (Dako) and employing the ECL detection system (Pharmacia). For detection of human ILRA protein, the antibody SC-25444 from Santa Cruz was used (1/200 dilution). As loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used (0.5 μg/ml, IMG-5143A, Imgenex).

Proteins were extracted, and lysates were analyzed as previously described in [9 (link)]. The GAPDH antibody IMG 5143A was purchased from Imgenex.

Whole-cell protein extracts were prepared from N2A and U251 cells, 20 hr after transfection with pCAG-DCC:TDTOMATO and pCAG-myr-TDTOMATO constructs (1 µg) as previously described (Bunt et al., 2010 (link)). Moreover, COS-7 protein extracts were prepared 48 hr after transfection with the pCAG-DCC:TDTOMATO wildtype and missense mutant receptor constructs (0.2 µg). Western blots were performed to detect mouse DCC expression levels using a goat polyclonal anti-DCC antibody (1:200 COS-7 or 1:800, sc-6535, Santa Cruz Biotechnology) and mouse NTN1 using a goat polyclonal antibody (1:500 U251 or 1:1000 N2A, AF1109, R&D Systems). GADPH was used as a loading control and detected using rabbit monoclonal anti-GADPH antibodies (1:2000, 2118, Cell Signaling Technology for COS-7; 1:1000, IMG-5143A, IMGENEX for N2A and U251).