Monoclonal anti gst

GST—Cyld and GST protein were both individually expressed in BL21(DE3) E. coli. Cleared lysates were prepared and the soluble fusion proteins were purified on glutathione Sepharose 4B GST-tagged protein purification resin (GE Healthcare Life Sciences,) and tested by immunoblot using monoclonal anti-GST (Cell Signaling Technology). His-tagged NLRP6 expressed by the pET-30a(+) vector was transformed into BL21(DE3) E. coli and soluble fusion proteins were purified on Ni-NTA His-binding resin (Thermo Fisher Scientific). Purified GST—Cyld and GST were individually incubated with glutathione beads for approximately 4 h. Then the beads were collected by centrifugation, and washed five times with GST pulldown lysis buffer and TBS (1:1). Then, purified His—NLRP6 protein was added to glutathione beads and incubated at 4 °C overnight with gentle rocking. The beads were washed five times and resuspended in SDS—SDS—PAGE sample buffer. After boiling, the bound proteins were analyzed on 12% SDS—PAGE gel followed by immunoblotting using anti-His. His—NLRP6 protein was used as a control.

RORγt and RORγt mutant (ΔS316A and ΔS316D) were sub-cloned into pGEX-6P1-DEST vector containing GST tag at N-terminal. Further, GST–RORγt and GST–RORγt mutant proteins were individually expressed in Rosetta^™(DE3) pLysS E. coli. Cleared lysates were prepared and the soluble fusion proteins were purified on glutathione Sepharose 4B GST-tagged protein purification resin (GE Healthcare Life Sciences) and tested by immunoblot using monoclonal anti-GST (Cell Signaling Technology). GST-Itch protein was purified as shown previously (Paul et al., 2018 (link)).