Figure 5.

HAUSP knock-down effect on the regulation of ANXA1 following UV-induced DNA damage. (a) Two different HAUSP-specific siRNAs as described in Materials and methods were transfected into HeLa cells. The cells were harvested and lysed 72 h after incubation. The depleted HAUSP levels were detected by western blotting using an anti-HAUSP antibody. (b) HeLa cells transfected with siRNA specific for HAUSP or control siRNA were exposed to UV and incubated for 3 h. Then, cell lysates were immunoprecipitated with an anti-ANXA1 antibody. Subsequent western blotting was performed to detect the ubiquitination level of ANXA1 using an anti-ubiquitin antibody. (c) HAUSP-depleted (HAUSP KD) and control shRNA transduced (Control) HeLa cells were generated by lentiviral induction as described in Materials and methods. Depleted HAUSP levels compared with normal HeLa cells were detected by western blotting using an anti-HAUSP antibody. (d) HeLa, HAUSP knock-down HeLa (HAUSP KD), and HAUSP overexpressed HeLa cells either incubated for 3 h following UV treatment or not were blotted with anti-HAUSP and anti-ANXA1 antibodies to detect the cleavage of ANXA1 protein. (e) UV-treated or normal HeLa KD cells were immunostained using FITC staining after incubation with an anti-ANXA1 antibody. DAPI was used for counterstaining of the nucleus (Green: ANXA1, and Blue: DAPI). (f) Nucleus-cytosol fraction was performed using HeLa and HeLa KD cell lysates. Then, HAUSP and ANXA1 levels were detected using anti-HAUSP and anti-ANXA1 antibodies. Anti-PARP and anti-α-tubulin antibodies were used as fraction controls. Statistical data are presented as a means (n=3, *P<0.05)