Total RNA of mice brain was extracted, CDS fragment of PICK1 and Orai1 was obtained by RT PCR, CDS were respectively sub cloned into pGEX 4T 2 with GST tag to generate GST PICK1 and sub cloned into pRK5 with myc tag to get myc Orai1, and recombinants were sequenced after confirmation by digested with SalⅠ and NotⅠ. Correct myc Orai1 vector was expressed in HEK293 cells, GST PICK1 vector was expressed in BL21 competent cells. GST PICK1 fusion proteins were analyzed with protein liquid mass spectrometry after being purified and cut SDS PAGE gel of the target band. And then GST PICK1 fusion protein and myc Orai1 fusion protein were mixed to apply GST pulldown experiment, and myc Orai1 band was detected by western blot. Result indicates PICK1 and Orai1 interacted in Vitro.
