Pflag cmv 1

Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Information of primary antibodies is as follows: mouse anti-FLAG (Sigma, F1804, 1:5000 for WB), mouse anti-GFP (Santa Cruz, sc-9996, 1:1000 for WB), mouse anti-GFP (Invitrogen, A-11,120, 1:1000 for staining), mouse anti-PSD95 (Millipore, MAB1598, 1:1000 for WB and 1:500 for staining), mouse anti-synaptophysin (Dako, M7315, 1:5000 for WB), rabbit anti-β-actin (Santa Cruz, sc-1616-R, 1:1000 for WB), mouse anti-Tau-1 antibody (Millipore, MAB3420, 1:500 for staining), mouse anti-MAP2 antibody (Millipore, MAB3418, 1:500 for staining), DGCR2 antibody was generated against hDGCR2-ICD in rabbit (1:1000 for WB and 1:200 for staining).
To generate FLAG-hDGCR2, the human DGCR2 cDNA encoding 22–550 amino acids of DGCR2 without signal peptide was amplified by PCR and subcloned into pFLAG-CMV1 (Sigma, E7273) downstream of an artificial signal peptide sequence and a FLAG epitope. Different cDNAs encoding ΔECD and ΔICD of DGCR2 were amplified with primers 5’- GAAGATCTGATGCGCCTGGTCGTC-3’ and 5’- ACGCGTCGACCTACACCACAGTATTG-3’, 5’-GAAGATCTGCGGCCAGAGCTG-3’ and 5’- ACGCGTCGACCTACCGGTGGACCATGAAG-3’, and subcloned into pFLAG-CMV1 separately. NRXNs and NLGNs constructs were obtained as described previously [48 (link)]. The authenticity of all constructs was verified by DNA sequencing and western blotting analysis.

We generated plasmids encoding various truncated mutants of UCP, pVHL and wild-type UbcH5c for expression in bacterial cells or mammalian cells [9 (link)]. For bacterial expression, plasmids were constructed by ligating PCR products into pET28a (Novagen, WI, USA) and pGEX-4T-1 (GE Healthcare Life Sciences, WI, USA), and for mammalian expression, pFlag-CMV1 (Sigma-Aldrich, MO, USA), pEBG and pcDNA3-HA (Invitrogen, CA, USA) were used. Mutants of UCP and VHL were generated based on the wild-type genes using a PCR method [16 (link)].