Nucleic Acids Research, 2008, Vol. 36, No. 19 6071 Figure 3. SUMO1-conjugated lysine 174 of LAP1 plays a functional role in the regulation of COX-2 transcription. (A) Western blot was performed with HA antibodies to detect A431 cell lysates from the transfectants of HA/LAP1, HA/LAP1K174, HA/LAP2 and HA/LAP2K151A with or without SUMO1 expression vectors. The bold arrowhead indicates the positions of suHA/LAP1 and suHA/LAP2. (B) A431 cells were transfected with expression vectors of HA/LAP1 or HA/LAP1K174A with or without SUMO1 siRNA for 24 h. The lysates of transfectants were harvested, and then for IP by SUMO1 antibody (Zymed). (C) The expression vectors of HA/LAP1 and HA/LAP2 were transiently transfected into A431 cells. After 6 h of starvation, these transfectants were stimulated with EGF and harvested at indicated times. The exogenous expressions of HA/LAP1 and HA/ LAP2 proteins were detected using HA antibodies. (D) A431 cells were transfected with COX-2 reporter combined with the expression vectors of HA/LAP1 or HA/LAP1K174A as indicated. Cell lysates were then prepared and analyzed for luciferase activity. The percentage of the repression activity of transfectants was normalized to the control transfectants of COX-2 reporter and pCDNA3/HA vector. P < 0.05; P < 0.01; P < 0.001. (E) A431 cells were transfected with COX-2 reporter and siRNA of CEBPB combined with the expression vectors of HA/LAP1 or HA/LAP1K174A as indicated. Cell lysates were then prepared and analyzed for luciferase activity. Values of EGF-treated cells compared to control cells were indicated. The percentage of the repression activity of transfectants (HA/LAP1, HA/LAP1K174A, HA/LAP2 and HA/LAP2K151A) was normalized to the control transfectants of COX-2 reporter and siRNA of CEBPB (left panel). Western blot was performed with CEBPB antibodies to conﬁrm the knockdown eﬃciency of CEBPB siRNA and verify the expression amounts of HA/LAP1, HA/LAP1K174A, HA/LAP2, HA/LAP2K151A and LIP (right panel). with various indicated expression vectors in A431 cells. Transfected CEBPB siRNA targeted the 30-UTR of CEBPBs mRNA, silencing the endogenous CEBPBs expression. Meanwhile, the CEBPB siRNA has no activity for these cotransfected expression vectors containing only the coding region of CEBPBs (Figure 3E, right panel). Consistent with Figure 3D, LAP1K174A can reverse the LAP1-mediated repression of COX-2 promoter activity. However, LAP2K151A shows a consistent COX-2 reporter activity compared with LAP2 upon EGF treatment (Figure 3E and Supplementary Figure S1). The results suggested that an intact lysine 174 of LAP1 is necessary for the repression of COX-2 promoter activity, and a sumoylation-independent mechanism of LAP2 is involved in the LAP2-mediated inactivation of COX-2 transcription.