Gfp rac1

Full-length cDNA of human CRACR2A-a (NCBI Reference Sequence: NM_001144958.1) was cloned from Jurkat cell cDNA library using primers described in Table S1 into pMSCV-CITE-eGFP-PGK-Puro vector with a N-terminal FLAG tag. The reverse primer was designed in the putative 3′ UTR region to sequence the endogenous STOP codon. Our clone contains an additional serine residue at amino acid position 425 within the proline-rich domain (codon 5′-AGT-3′). Various mutants of CRACR2A-a were generated by PCR amplification and site-directed mutagenesis using primers described in Table S1. CRACR2A-a plasmids were N-terminally tagged with a FLAG tag and subcloned into a lentiviral vector, FGllF (kind gift from Dr. Dong Sun An, UCLA). The cDNAs encoding WT and mutants of CRACR2A-a were also subcloned into pC1-mCherry and pEGFP-C1 vectors (Clontech) to generate N-terminally mCherry or GFP-fused proteins. HEK293 and Jurkat E6-1 T cell lines were obtained from American Type Culture Collection center (ATCC, Manassas, VA). Raji B cells were a kind gift from Dr. Sherie Morrison’s laboratory (UCLA). Vav1-GFP, ZAP70-GFP and GFP-Rac1 clones were purchased from Addgene. LAT1-CFP clone and J.Vav1 cells were a kind gift from Dr. Larry Samelson’s laboratory (NIH). LAT1 cDNA was excised out using EcoRI and BamH1 sites and subcloned into pEGFP-N1 vector.