LGR4, ZNRF3

Getting: PCR

Connecting: Overlapping

V5-tag

Signaling peptide

Linker length

Fc tag adding

Preparation:

Transfection and collection (HEK293)

RTAC (V5-tagged)

ECD (ZNRF3, LGR4; V5-tagged)

4 (HA-tagged)

Secretion testing

Stability testing

Affinity testing

Anti-cancer testing

(HEK293, RKO)

Western Blotting (HEK293, RKO)

RT-PCR (HEK293, RKO)

MTT (RKO, U2OS, Patu8988s)

Preparation:

Gene knockout: ste2, sst2 and selection

PCR

P2Y2

Chimeric Gα

P2A

P2Y2-P2A-mCherry

P2Y2-P2A-Gα

pFUS1-mCherry

pFUS1-RTAC

P2Y2-P2A-Gα yeast selection

P2Y2-P2A-Gα+pFUS1-RTAC yeast selection (R-yeast)

Co-culture (RKO)

Western Blotting

RT-PCR

MTT

pHIS3-NeoR-pCUP1-HSF1

R-yeast with the kill switch selection

Spotting assay