BMC Cancer 2006, 6:109 http://www.biomedcentral.com/1471-2407/6/109 FWigeustreern1blot for testing the specificity of prepared rabbit KIAA0101 antibody Western blot for testing the specificity of prepared rabbit KIAA0101 antibody. A: the recombinant pEGFP+0101 and pEGFP plasmids were transfected into cells separately. 48 hours later, the transfected cell protein were extracted for Western blot analysis with GFP antibody (1:1000) and prepared rabbit KIAA0101 antibody (1:1000) respectively. B: the KIAA0101 prokaryo- tic expression protein was loaded at different quantity (10 ng, 5 ng and 2 ng) and reacted for the test of the prepared KIAA0101 antibody specificity. RIPA buffer, followed separated by 15% SDS-PAGE elec- trophoresis and for the KIAA0101 Western blot. Co-transfection and separation of transient expression cells Transfection was performed by incubating cells with pcDNA3.1/A+0101 together with the pMACS KKII plas- mid (5:1) (Miltenyi Biotec, Auburn, CA, USA). pMACS KKII plasmid could be provided for the magnetic isolation of transiently transfected mammalian cells. Briefly, 4*106 cells in 6-cm plate were incubated for 5 hours at 37°C with 5 ug of the plasmid pcDNA3.1/A+0101 and 1 ug of pMACS KKII magnetic plasmid in a total of 1 ml DMEM. The cells were then washed and incubated overnight in DMEM containing 10% FCS at 37°C in 5% CO2 atmos- phere. 48 hours later, the cells were labeled by MACSselect KKII microbeads and selected by MACSselect KKII column according to the protocol recommended. At the same time, the co-transfection of pcDNA3.1/A with pMACS KKII plasmids into cells is performed as control. Cell growth analysis KIAA0101 transiently expression positively (+) cells iso- lated by the MACSselect KKII microbeads and negative cells were seeded into 96-well flat-bottom microtiter plates equally. Cells were cultured in DMEM containing 10% FBS. Each day, 10 ul of CKK-8 solution (Dojin, Osaka, Japan) were added to each well and incubated for 2 hrs at 37°C. Optical density was read at 450 nm in a microplate reader. Cell cycle analysis KIAA0101 transiently expression positively (+) cells iso- lated by the MACSselect KKII microbeads and negative cells were fixed in PBS/70% EtOH at -20°C overnight. On the following day, cells were washed in PBS, stained with propidium iodide (50 mg/ml) and RNAse (360 mg/ml) for 30 min at 37°C, then with just propidium iodide (50 mg/ml) in PBS for at least 1 h at 4°C. DNA content was quantified using a FACSCAN (Becton Dickson). This experiment was repeated three times. Results Preparation of polyclonal antibody against KIAA0101 and test of its specificity As the antibody for KIAA0101 was not commercial avail- able, we prepared the polyclonal rabbit antibody against full-length KIAA0101-His fusion protein. The pEGFP+0101 recombinant plasmid was constructed and transfected into HCC cells. The proteins of the transfected cells were extracted for Western blot by using the anti-GFP antibody and KIAA0101 rabbit antibody to test the expression of EGFP and KIAA0101. As illustrated in Fig 1, a band of EGFP-KIAA0101 fused protein could be revealed by both the anti-GFP and anti-KIAA0101 anti- bodies. On the contrary, no band was observed for pEGFP vector-transfected cell protein by anti-KIAA0101 anti- body, though it was positive by anti-GFP antibody. Also we used the KIAA0101 prokaryotic expression for the test of its antibody specificity. The KIAA0101 antibody could detect a specific band with the predicted size (15 KD) and Page 3 of 8 (page number not for citation purposes)