Fig. 4. Expression of HERC5 protein. (A) HERC5 protein expression. Immunoprecipitation with anti-HERC5 serum from HSMECs, HUVECs and HSMCs metabolically labeled for 16 hours were analyzed by 9% SDS-PAGE. Samples are normalized to cell number. HERC5 protein is marked by an asterisk. (B) Immunoprecipitation with anti-HERC5 serum from HSMECs metabolically labeled for 16 hours in the absence or presence of LPS were resolved by 6.5% SDS-PAGE. Duplicates were immunoprecipitated with pre-immune serum as controls. Integrity of ECs was tested by IL-8 secretion upon proinflammatory cytokine treatment before assaying. Cytosolic cell extracts are normalized to counts. HERC5 signal is marked by an asterisk. pi-serum, pre-immune serum. (C) Results from pulse-chase experiments in HSMECs in the absence () or presence () of LPS during chase. The calculated regression curves were significantly different to each other (P<0.05). (D) HERC5 protein was precipitated from cell lysates of HSMECs metabolically labeled for 20 hours. Where indicated, LPS was added for 8 hours and CHX for 4 hours before cell lysis. As a control, actin was precipitated from the same protein extracts. CHX, cycloheximide.