Figure 1

Validation of an ‘in-house’-generated anti-PAT4 monoclonal antibody. (a and b) Formalin-fixed, paraffin-embedded 786-O cells incubated with PAT4 monoclonal antibody (Pat4/9/H10) and visualised with 3,3'-diaminobenzidine (DAB). There is an obvious perinuclear region of staining visible in most cells treated with the scrambled control siRNA (a). This staining is absent when the PAT4 transcript is knocked down using an siRNA against PAT4 (si435; Heublein et al.;¹⁴ b). (c) Western blot analysis of serial dilutions of cell lysates (15, 7.5 and 3.8 μg of protein) produced from pools of 786-O cells carrying an IPTG-inducible shPAT4 (43587; shPAT4) probed with PAT4 monoclonal antibody Pat/9/H10. This reveals a set of bands from 60–75 kDa that is strongly reduced by IPTG-induced PAT4 knockdown (+IPTG), suggesting that they are PAT4-specific. Western blots were also probed with an anti-tubulin antibody as a loading control. (d) Western blot of cell lysates from 786-O cells and from a GFP-PAT4-overexpressing HEK-293 cell line treated with PNGase F before electrophoresis to remove glycosyl groups. This resolves the crossreacting molecules seen in untreated cell lysates into more specific bands migrating at ~30 and 50 kDa, respectively, smaller than the predicted molecular weights of 55 kDa (PAT4) and 85 kDa (GFP-PAT4), a phenomenon also reported for other transmembrane proteins.⁴³