Table 2. Site-directed mutagenesis primers for SNAP/FLAG-hGLP-1R cholesterol binding mutant generation.

GLP-1R cholesterol binding mutant	Forward primer (5’–3’)	Reverse primer (5’–3’)	Side chain charge change
BATCH 1 SCREEN
I323A	CATTGGGGTGAACTTCCTCGCCTTTGTTCGGGTCATCTGC	GCAGATGACCCGAACAAAGGCGAGGAAGTTCACCCCAATG	non-polar to non-polar
S163A	CTCTGGTTATCGCCGCTGCGATCCTCCTC	GAGGAGGATCGCAGCGGCGATAACCAGAG	polar to non-polar
S225A	CCAGGACTCTCTGGCCTGCCGCCTGGTG	CACCAGGCGGCAGGCCAGAGAGTCCTGG	polar to non-polar
V229A	GAGCTGCCGCCTGGCGTTTCTGCTCATGC	GCATGAGCAGAAACGCCAGGCGGCAGCTC	non-polar to non-polar
BATCH 2 SCREEN
L167A	CCTCTGCGATCCTCGCCGGCTTCAGACACC	GGTGTCTGAAGCCGGCGAGGATCGCAGAGG	non-polar to non-polar
V160A	CCTTCTCTGCTCTGGCTATCGCCTCTGCGAT	ATCGCAGAGGCGATAGCCAGAGCAGAGAAGG	non-polar to non-polar
I165A	GTTATCGCCTCTGCGGCCCTCCTCGGCTTCAG	CTGAAGCCGAGGAGGGCCGCAGAGGCGATAAC	non-polar to non-polar
G151A	CATCTACACGGTGGCCTACGCACTCTCCT	AGGAGAGTGCGTAGGCCACCGTGTAGATG	non-polar to non-polar
BATCH 3 SCREEN
D372A	GCCTTTGTGATGGCCGAGCACGCCCGG	CCGGGCGTGCTCGGCCATCACAAAGGC	acidic to non-polar
H363A	CATCCCCCTGCTGGGGACTGCTGAGGTCATCTTTG	CAAAGATGACCTCAGCAGTCCCCAGCAGGGGGATG	basic to non-polar
L401A	AGGGGCTGATGGTGGCCATAGCATACTGCTTTGTCAACAAT	ATTGTTGACAAAGCAGTATGCTATGGCCACCATCAGCCCCT	non-polar to non-polar
I400A	CAGGGGCTGATGGTGGCCGCATTATACTGCTTTGTCAAC	GTTGACAAAGCAGTATAATGCGGCCACCATCAGCCCCTG	non-polar to non-polar