The Human GPR35a coding sequence was obtained from RefSeq entry NM_005301. Primers with Eco RI and Bam HI (1 and 2 in table S2, respectively) linker sequences were used to amplify the GPR35 coding sequence from human cDNA. After double digestion of the resulting PCR fragment with Eco RI and Bam HI, the DNA was cloned into the pEXPR-IBA103 Human Expression Vector (IBA Lifesciences, Goettingen, Germany). This resulted in a C-terminal TwinStrep-tagged GPR35 expression system.

We generated an identical construct for the GPR35aT108M variant through site-directed mutagenesis. First, two overlapping PCR fragments were generated using the mutagenic primers separately (3 and 4 in table S2), combined with the previously mentioned Eco RI/Bam HI linker primers (1 and 2 in table S2, respectively). In a second step, full-length GPR35a coding sequence was generated with the linker primers (1 and 2 in table S2) only. The resulting fragment was cloned into pEXPR-IBA103.

Constructs for FRET experiments were generated using vector backbones mVenusN1 and mCeruleanN1 that were gifts from S. Vogel (Addgene plasmids 27793 and 27795). We generated GPR35a and GPR35aT108M PCR fragments with Eco RI/Bam HI linker sequences using the primer pair 5 and 6 (table S2) for C-terminal fusion. Resulting PCR fragments and vector backbones were digested with Eco RI/Bam HI. ATP1A1 cDNA (RefSeq entry NM_000701.7) was amplified using the primer pair 7 and 8 (table S2) for C-terminal fusion. PCR fragments of ATP1A1 were cloned after Xho I/Xma I digestion. CXCR2 cDNA was amplified using primers 9 and 10 (table S2), and CCR5 was amplified using primers 11 and 12 (table S2). Both cDNAs were cloned after Xho I/Hind III digestion. We used N-terminal HA-tagged expression vectors for GPR35 (GPR035TN00), CXCR2 (CXCR20TN00), CCR5 (CCR050TN00), and P2Y12R (P2Y120TN00) obtained from the cDNA Resource Center (Bloomsburg University, USA). Myc-DDK–tagged expression vectors for ATP1A1 (RC201009) and ATP1A3 (RC203198) and untagged expression vector for ATP1A2 (SC119715) were obtained as lyophilized DNA (OriGene, Rockville, MD). ATP1A2 cDNA was amplified using primers 13 and 14 (table S2), digested with Sgf I/Mlu I, and ligated into predigested pCMV6-Entry. All expression vectors were cloned and stored in DH5alpha. All constructs were verified by Sanger sequencing.

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