293t 17 hek 293t 17

CHO-K1 cells stably expressing SNAP-GLP1R (CHO-K1_SNAP-GLP1R) were generated by and obtained from Dr Ben Jones, Imperial College London, UK. HEK 293 and HEK 293T cells were purchased from ATCC: 293 [HEK-293] (ATCC® CRL-1573™) and 293T/17 [HEK 293T/17] (ATCC® CRL-11268™). All cell lines were regularly tested for mycoplasma.

HEK293T cells(293T/17 [HEK 293T/17] (ATCC® CRL11268™)) were cultured in DMEM medium with supplement of 10% FBS (GIBCO) and 1% Penicillin-Streptomycin stock solution (Invitrogen, 5,000unit Penicillin and 5,000 μg Streptomycin per milliliter). The expression vectors of HA/V5-tagged A3B and A3DE were constructed in our laboratory. HA-tagged A3DE-E80Q, A3DE-E264Q, A3DE- E80Q/E264Q and A3B-W228L/ D316N were generated by overlapping PCR and confirmed by sequencing. The human L1 plasmids 99 PUR L1RP EGFP (L1) and 99 PUR JM111 EGFP (JM111, an L1 construct containing two point mutations in ORF1, which cause complete abolishment of LINE-1 retrotransposition, was used as a negative control) were gifted from Professor Kazazian HH Jr and had been described previously [40 (link)]. The pcDNA3.1-EGFP plasmid was cloned in our laboratory.

293T/17 [HEK 293T/17] (ATCC CRL11268) were purchased from AMERICAN TYPE CULTURE COLLECTION (ATCC) (lot number 58483269, cell species identity has been confirmed by COI assay (intraspecies) and STR assay (interspecies); cells tested negative for mycoplasma). Cells were cultured to 60–70% confluence in 10% FBS-DMEM. Expression plasmids (HA-ARPP-16, HA-S46D-ARPP-16, HA-S88D-ARPP-16, HA-MAST3 [aa from 332 to 1014], HA-T389D-MAST3, HA-T389A-MAST3, HA-S512D-MAST3, HA-S512A-MAST3, HA-T628A-MAST3, HA-T628D-MAST3 and HA-S747A-MAST3 were expressed in HEK293T cells in 10 cm plates using Lipofectamine 2000 transfection (Invitrogen). Immunoblotting for ARPP-16 or MAST3 was routinely used to control for levels of protein expression. Expression levels of ARPP-16 plus endogenous ARPP-19, or MAST3, were approximately equal to that of ARPP-16 plus ENSA, or MAST3, in adult striatum. Twenty-four hours after transfection, cells were lysed in a buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X-100, protease and phosphatase inhibitors cocktails, followed by centrifugation at 16,000 x g, 10 min. Supernatants were subjected to immunoprecipitation or immunoblotting as described below.