Fig. 2.

H2AX, 53BP1 and MDC1 promote phosphorylation of SMC1 and SMC3 following irradiation. (A) Western blot for H2AX, 53BP1 and MDC1 following siRNA depletion. HeLa cells were transfected with the indicated siRNAs (Supplementary Table S1), incubated for 48 h and X-irradiated with 10 Gy. Cells were harvested after 1 h to allow repair, 20 μg was loaded per lane and actin was used as loading control. (B) Western blot to detect SMC1pS966 and actin in 20 μg of extracts prepared in (A). SMC1pS966 signals were quantified using the Kodak Molecular Imaging software V4.0.1 and normalized to the actin signals (bar diagram). Total SMC1 signals are shown to ensure that SMC1 was present after each siRNA treatment. (C) Immuno-precipitation of SMC3pS1083 in 100 μg of extracts prepared in (A). pc = positive control. SMC3pS1083 signals were quantified as in (B) and normalized to the antibody bands (bar diagram). Error bars represent standard deviations of 2 independent experiments. The Student's t-test was used to compare lacZ siRNA-treated control to any other sample. P values between 0.01 and 0.05 are marked by ‘*’ and between 0.001 and 0.01 by ‘**’. Total SMC3 signals are detected on a Western blot run with the same extracts used for IP to show that SMC3 is present in the extracts.