Gfp actin

For Myc-tagged AF expression plasmid, full-length rat AF (residue 1-640), N-terminal fragment AF-N (residue 1-300), and C-terminal fragment AF-C (residue 301-640) were PCR-amplified and subcloned into vector GW1-Myc2b digested by BglII and EcoRI. To generate AF-miR knockdown constructs, miRNA was designed using BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress/). According to the ranking score, the top two miRNAs (nucleotide residues 590-610 and nucleotide residues 709-729) were chosen, linked together, and cloned into pcDNA6.2-GW/EmGFP vector. Accordingly, this knockdown plasmid expresses two AF miRNAs, resulting in better knockdown efficiency. A control vector (pcDNA6.2–GW/EmGFP-miR, Invitrogen) expressing a miRNA predicted to not target any gene in mammalian genomes was used as the negative control in knockdown experiments. To generate the silent mutant resistant to AF-miR, site-directed mutagenesis was performed using the following oligonucleotide: 5′-GATTTGCTGATACGCACTCAT-3′ for nucleotide residues 590-610 and 5′-CTGGAACTTGTATCTAGTGAT-3′ for nucleotide residues 709-729, respectively. The underlined bases indicate the mutated sites. GFP-actin was purchased from Clontech and used to outline neuronal morphology.

SDC2, SDC2ΔC2, SDC2 RNAi knockdown, FGF22-mCherry, FGF22-GFP and FGF22 RNAi knockdown constructs have been previously described (Umemori et al.24 (link); Lin et al.18 (link); Terauchi et al.25 (link); Terauchi et al.26 (link)). For myc-tagged FGF22, full-length FGF22 cDNA was subcloned into the GW1-myc vector. To generate the silent mutant resistant to sh-FGF22, site-directed mutagenesis was performed using the following oligonucleotide: 5′-GGCAAGGGAGACGGACTCGACGGCATCAA-3′ (bases in italics indicate the mutated sites). For CD8T-SDC2C fusion, the cDNA fragment corresponding to the first 220 amino acid residues of mouse CD8α was PCR amplified and fused with the fragment containing the last 32 amino acid residues of SDC2. An extra BglII site was inserted between the fragments of CD8α and SDC2 for cloning purposes. GFP-actin was purchased from Clontech.