BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 Additional file 2 Details of open reading frame selection, microarray oligonucleotides, qRT-PCR primers, database annotation and prior work. Functional groups assigned based on likely orthologs and/or domain annotation from multiple sources. Accession numbers are for T. brucei 927 at GeneDB, except ESAG6 and ESAG7 which are for T. brucei 427 at GeneDB, and VSG oligonucleotides which are based on sequences deposited at NCBI. All oligonucleotide sequences are given in 5'-3' orientation. Array version 1.0 was used for the bloodstream v procyclic comparison and analysis of the Rab5 overexpressor lines; array version 1.1 used in all other experi- ments. The "Cross-hybridising ORFs" column gives the GeneDB accession numbers of ORFS likely to cross-hybridise to each oligonucleotide, when applicable, as predicted by the OligoArray 2.0 program. qRT-PCR oligo- nucleotide sequences are given for all ORFs analysed by qRT-PCR. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-298-S2.xls] Additional file 3 Developmental regulation of components of the adaptin complexes AP1, AP3, and AP4 in T. brucei. Panel A: Heatmap of bloodstream ver- sus procyclic form signal ratios for the ORFs corresponding to the adaptin complexes, based on eight microarray experiments. For each target gene, the four replicate spots on the array were averaged, following removal of inconsistent replicates. The scale shows the colour scheme for the z-score of the data, indicating how far and in what direction, the ratio for each spot deviates from the mean for each array, expressed in units of standard deviation; bright red indicates significant upregulation in BSF, bright green indicates significant upregulation in PCF, dark colours or black indicate no differential expression between the two developmental stages. AP1σ is highly upregulated in BSF, and AP3μ in PCF (also see Table 1) with a general trend of BSF upregulation for AP1 components and PCF upregulation (or BSF downregulation) for AP3 and AP4 components. Panel B: Western blot analysis of whole cell lysates from BSF (lane B) and PCF (lane P) (1 × 107 cell equivalents) probed with rabbit antisera raised against recombinant TbδAd adaptin subunit (AP3). The scale to the left represents relative molecular mass in kDa. The predicted molecular weight for the AP3δ protein is 125 kDa. Loading equivalence was monitored by Ponceau Red staining of the membrane after transfer (not shown). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-298-S3.pdf] Additional file 4 Response of T. brucei cells to dithiothreitol and tunicamycin treat- ment. Panel A: Growth curves for BSF cultures after the addition of dithi- othreitol (DTT, 1–10 mM final concentrations) or tunicamycin (5–10 μg/ml final concentrations). Cell numbers diminish rapidly after the addi- tion of DTT (within 4 hours), whereas cell growth is arrested after addi- tion of tunicamycin and cell numbers remain stable for up to 24 hours. Panel B: Western blot analysis of whole cell lysates (1 × 107 cell equiva- lents) for BiP in a control culture, as well as cultures supplemented with 1 mM DTT and 5 μg/ml tunicamycin. A culture of BSF cells was divided; one flask was retained as control and DTT or tunicamycin was added to the other subcultures. Samples were taken prior to DTT or tunicamycin addition (C0), 1 hr and 4 hr after DTT treatment (D1 and D4, respec- tively), 1 hr, 4 hr and 24 hr after tunicamycin treatment (T1, T4 and T24), and at 1 hr, 4 hr and 24 hr from the control (C1, C4, C24). Panel C: BiP protein levels normalised to total protein, based on the Western blot in panel B and two additional replicates. The levels of BiP antigen are nor- malized to 100% for the control culture at time zero (C0). Panel D: Rel- ative levels of BiP mRNA as measured by qRT-PCR after DTT (black bars) and tunicamycin (grey bars) treatment. The levels of BiP mRNA are normalized to 100% for the control culture at time zero (C0). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-298-S4.pdf] Additional file 5 Verification of phenotypes for VSG and CLH RNAi cell lines. Panel A: Growth curves of induced versus uninduced VSG RNAi cell lines, indicat- ing growth arrest in the induced cell line over four days post induction. Panel B: CLH protein levels in induced (+Tet) versus uninduced (-Tet) CLH RNAi cell lines were examined by Western blotting. BiP was used as a loading control. Data from two biological replicates are shown. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-298-S5.pdf] Additional file 6 Significantly developmentally expressed trypanosome genes grouped by functional class. Numbers of transcripts on which the graph in Figure 2 is based. The number of genes for each functional class correspond to the ORFs featured in Table 1 and discussed in the text. Note that several of the oligonucleotides on the array target multicopy genes (e.g, tubulin, his- tones) and thus the total number of ORFs subject to differential regulation is higher than the number of ORFs/oligonucleotides given in Table 1. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-298-S6.pdf] Acknowledgements We are grateful to the following for assistance with ORF selection: Amanda O'Reilly, Carme Gabernet-Castello, Sara Melville, Mark Carrington and Jer- emy Mottram. We also thank Sergio Callejas and Anthony Brown for assist- ance with array design, Krystyna Kelly and Rob Furlong for advice with data analysis and Karl Hoffmann and Jennifer Fitzpatrick for help with qRT-PCR. Special thanks to Clare Allen for production of the antibody to TbδAd and to Derek Nolan for providing mRNA from MITat1.1 trypanosomes. We also thank Joel Dacks and Mark Carrington for comments on the manu- script. This work was supported by program and project grants from the Wellcome Trust (to MCF). The microarray designs used in this study as well as the relevant experimental data have been deposited in ArrayExpress Page 24 of 28 (page number not for citation purposes)