The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells
Figure 2
The over-expression of the β2 subunit of proteasome decreases HR. To determine if the β2 subunit was over-expressed in HeLaG1 cells, first we carried out Western blot analysis of total protein extracts 24 hours after transfection (a) and in the stably clonal population transfected with the β2 plasmid (b) using the anti-myc as primary antibody to detect the myc-tagged β2 subunit. The endogenous myc is indicated in (a) and (b) and is used as loading control. In (c), we carried out Western blot using the anti-PSMB2 (β2 subunit) as primary antibody. 30 μg of total protein extract was loaded as follows: lane 1, total protein extract from cells transfected with the plasmid expressing the β2 subunit; lane 2, total protein extract from cells transfected with the plasmid pcDNA6myc. Intrachromosomal HR was determined 24 hours after the transfection of the HeLa G1 with the plasmid expressing the β2 subunit or with the empty pcDNA6myc (d); recombination was also measured in the cell population derived from HeLaG1 stably transfected with the plasmid expressing the β2 subunit or with the empty pcDNA6myc (e). The frequency of HR was calculated by dividing the total number of HYGR colonies by the number of viable cells. Data are reported as mean of five independent experiments ± standard deviation. Results are statistically analyzed using Student -test. .
