Pegfp tagged hsp70i

A pEGFP-tagged Hsp70i was (plasmid 15215; Addgene) used in the FLECS assay and was originally cloned by Evan Eisenburg (Zeng et al., 2004). ATP 200 mM stock was prepared with low salt buffer. FuGENE 6 transfection reagent (Roche) was used for transfection of GFP-Hsp70i into HEK293T cells, following the manufacturer protocol. The transfection ensued for 48 hr, upon which time the cells were harvested and lysed in cell lysis buffer and one tablet Complete Mini protease inhibitor [Roche]). Cell lysates were stored at −80°C until further use. After binding, the resin lysates were washed three times with high stringency wash buffer and three times with low-stringency wash buffer. Next, the resin with bound proteins (50μl) were transferred to 0.2 μm polyvinylidene fluoride filter 96-well plate (Corning) sitting on top of a black flat-bottomed 96-well catch plate (Corning). Inhibitors or ATP were added to each well (50μl) and the plates were centrifuged using an Eppendorf Centrifuge5810 at 2,000 rpm for 2 min.

A pEGFP-tagged Hsp70i was (plasmid 15215, Addgene, Cambridge, MA) used in the FLECS assay and was originally cloned by Evan Eisenburg (Zeng et al., 2004 (link)). ATP used in the assay was purchased from Sigma (St. Louis, MO) and a 200 mM stock was prepared with low salt buffer (150 mM NaCl, 25 mM Tris, pH 7.5, 60 mM MgCl2). The γ-phosphate ATP sepharose was synthesized as previously described and stored in low salt buffer (Carlson et al., 2013 ). FuGENE 6 transfection reagent (Roche, Mannheim, Germany) was used for transfection of GFP-Hsp70i into HEK 293T cells, following the manufacturer protocol. The transfection ensued for 48 hours, upon which time the cells were harvested and lysed in cell lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, and 1 tablet Complete Mini protease inhibitor(Roche)). Cell lysates were stored at −80°C until further use. Upon binding the resin lysates were washed 3× with high stringency wash buffer (1 M NaCl, 25 mM Tris, pH 7.5, 60 mM MgCl2, 1 mM DTT) and 3× with low stringency wash buffer (150 mM NaCl, 25 mM Tris, pH 7.5, 60 mM MgCl2, 1 mM DTT). Next the lysates were transferred to 0.2 µm PVDF filter 96-well plate (Corning, Corning, NY) sitting on top of a black flat-bottomed 96-well catch plate (Corning). The plates were spun down using an Eppendorf Centrifuge 5810 (Hamburg, Germany) at 2000 rpm for 2 min.