Anti smc3

Published, and commercially available antibodies used in this study are as follows: rabbit polyclonal anti-ATM (Abcam), rabbit polyclonal anti-SMC1A (Bethyl), rabbit polyclonal anti-SMC3 (Bethyl), rabbit polyclonal anti-RAD21 (Bethyl), rabbit polyclonal anti-Actin (Bethyl) and mouse monoclonal anti-Tubulin (Sigma-Aldrich). In addition, we generated a rabbit polyclonal antibody against the fragment 308–432 (Supplementary Table S1) amino acids of SMC1B protein. The fragment 308–432 was subcloned into Escherichia coli containing the plasmid p2N. The His-tagged protein was over-expressed and purified by Immobilized Metal Chelating Chromatography (IMAC) in denaturing buffer, a robust method for purifying histidine-tagged recombinant proteins. Rabbits were boosted subsequently 4 times with protein (200 μg/rabbit) mixed with IFA at 2-weeks interval. After 3 boostings, polyclonal antibody serum was tested by enzyme-linked immunosorbent assay (ELISA).

When appropriate cells were pre-extracted with ice-cold 0.2% triton solution in PBS 1× for 1 min and fixed for 20 min in formaldehyde solution 4%. If the extraction protocol was not carried out, cells were permeabilised for 4 min in 0.2% triton solution in PBS 1× after fixation. Cells were blocked in blocking solution (1% BSA, 0.2% Tween in PBS) for 1 h at room temperature. The incubation with the primary antibodies anti-RPA2 (Millipore RPA34-20 1:500), anti-Phospho-Histone H2A.X (γH2AX) (Ser139) (Cell Signaling Technology g-H2AX 20E3 1:250), anti-SMC1 (Bethyl laboratories A300-055A 1:1000), anti-SMC3 (Bethyl laboratories A300-060A 1:2000), anti-Mcm7 (Santa Cruz Biotechnology sc-56324 1:150), anti-RPA32 Phospho S4/S8 (Bethyl laboratories A300-245A 1:500) and anti-GFP (Abcam AB1218 1:1000) was carried out overnight. The following day the coverslips were incubated with secondary antibodies (Alexa Fluor® 488 goat anti-mouse 1:2000 and Alexa Fluor® 647 goat anti-rabbit 1:2000) for 2 h at room temperature. The cells were stained with Hoechst (Invitrogen) solution 1:10,000 in PBS 1×. The coverslips were mounted on slides with mounting medium Fluoroshied (Sigma). Images were obtained with Leica SPE2 40x objective lens and processed with Fiji.
For the quantitative analysis, between 200 and 300 cells were analysed per sample.