Fig 3. HSC70 and HSP70 association with proteins lacking binding partners.

(A) Top: schematic diagram of the Mre11/Rad50/Nbs1 complex showing that loss of Mre11 induces loss of Rad50. HSP70 and HSC70 UBAIT isolations were performed with or without depletion of endogenous Mre11 by shRNA (N = 6). Levels of Rad50 associated with HSP70 or HSC70 UBAITs were measured using label-free quantification. Western blot showing levels of Mre11, Rad50, and control proteins GAPDH and beta-actin in UBAIT cells with Mre11 shRNA, as indicated. (B) Top: schematic diagram of the ATP1A1/ATP1B1 complex showing that insufficient levels of ATP1B1 lead to loss of ATP1A1, while overexpression of ATP1B1 restores the complex and ATP1A1 stability. HSP70 and HSC70 UBAIT isolations were performed with or without overexpression of ATP1B1 (N = 6). Levels of ATP1A1 associated with HSP70 or HSC70 UBAITs were measured using label-free quantification. Western blot showing levels of ATP1A1, ATP1B1, and beta-actin in UBAIT cells with ATP1B1 expression, as indicated. (C) Top: schematic diagram of the MSH2/MSH6 complex showing that insufficient levels of MSH2 lead to loss of MSH2, while overexpression of MSH2 restores the complex and MSH6 stability. HSP70 and HSC70 UBAIT isolations were performed with or without overexpression of MSH2 (N = 6). Levels of MSH6 associated with HSP70 or HSC70 UBAITs were measured using label-free quantification. Western blot showing levels of MSH2, MSH6, and beta-actin in UBAIT cells with MSH2 expression, as indicated. See also S6 Data. ATP1A1, ATPase, Na+/K+ transporting subunit alpha 1; ATP1B1, ATPase, Na+/K+ transporting subunit beta 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSC, heat shock cognate; HSP, heat shock protein; Mre11, Meiotic recombination 11 protein; MSH, MutS homolog; Nbs1, Nijmegen Breakage Syndrome protein 1/Nibrin; shRNA, small hairpin RNA; UBAIT, ubiquitin-activated interaction trap; WT, wild-type.