BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 A B C 100000 10000 1000 e 100 c n e 10 c s reo 1 u lf5 10000 y C 1000 100 10 1 1 d lfo 20 / s l e v le 15 n o i s s e rxp 10 e A N Rm 5 e v i t a le 0 R BSF WT PCF 5AWT 5AQL 5BQL WT CLH BiP Rab5A BSF v PCF 5AWT v WT 5AQL v WT 5BQL v WT 10 100 1000 10000 1 10 100 1000 10000 100000 Cy3 fluorescence Cell line: SMB PCF Rab5AWT Rab5AQL Rab5BQL Rab5A Rab5B CLH AK3 COPIε RabX3 Open reading frame analysed LFiimguitreed 5differential expression in Rab5-overexpressing PCF cells Limited differential expression in Rab5-overexpressing PCF cells. Panel A: Western blot analysis for the clathrin heavy chain (CLH) and Rab5A, for wildtype BSFs and PCFs, PCFs overexpressing Rab5A (5AWT), Rab5AQL (GTP-locked mutant) and Rab5BQL (GTP-locked mutant) cell lines. BiP was used as a loading control. Panel B: Scatter plots of raw data for representative microarrays for BSF (Cy5) vs PCF (Cy3) experiment (plot as in Figure 1 for comparison), and Rab5-overexpess- ing lines (Cy5) vs PCF (Cy3) as indicated. Cy5 fluorescence is plotted on the Y-axis and Cy3 fluorescence on the X-axis. Spots with a Cy5/Cy3 or Cy3/Cy5 ratio above two are highlighted in black, spots with a ratio below two are shown in grey. Panel C: Relative expression levels of Rab5A, Rab5B, CLH, RabX3, COPIε, and adenylate kinase 3 (AK3) in wildtype SMBs and PCFs, and in the Rab5-overexpressor PCF lines, as assessed by qRT-PCR. Page 17 of 28 (page number not for citation purposes)