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A
Quantitative analysis of RAD51 binding to MMS22L–TONSL. Proteins were mixed in the indicated ratio, the MMS22L–TONSL complex was retained on amylose beads, and proteins in the eluate were analyzed by silver staining. Lane 6, the same amount of RAD51 was used as in lane 5, but no MMS22L–TONSL (MT) was added to control for non‐specific binding of RAD51 to the resin.
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B
NiNTA pulldown of his‐tagged MMS22L and RAD51. Proteins in the eluate were detected by silver staining.
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C, D
Quantitative analysis of RAD51 binding to MMS22L. Proteins were mixed with the indicated ratio, MMS22L immobilized on NiNTA beads and the eluate analyzed by silver staining. Lane 6: the same amount of RAD51 was used as in lane 5, but no MMS22L (No M) was added to control for non‐specific binding of RAD51 to the resin. Proteins in the eluates were quantified by comparing with known amounts of MMS22L or RAD51, respectively. Averages shown, n = 2; error bars, SEM.
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E
Purification of the MMS22L FA‐TONSL complex. The MMS22L FA mutant was co‐expressed with TONSL and the complex purified from Sf9 cells using the same protocol as for wild‐type MMS22L–TONSL. Although wild‐type and mutant heterodimers purified with approximately 1:1 stoichiometry, the yield of the mutant heterodimer was dramatically lower.
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F–H
Representative microscopy images of DNA damage‐induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA‐resistant variants of HSS‐MMS22L treated with 50 nM CPT for 18 h (F, G) or 5 μM ETP for 1 h followed by 3‐h incubation without the drug (H). Proteins are stained using indicated antibodies. Scale bars: 5 μm.
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I, J
Western blot analysis of cell extracts from cells used in representative experiments described in Figs
2E,
5H–K and
EV6F–H. Tubulin serves as a loading control.
