Dh5α competent cells

The Lin28a open reading frame was PCR-amplified from pMSCV-mLin28A using a forward primer that incorporated a 5′ EcoRI restriction site (5′-CGGAATTCATGGGCTCGGTGTCCAACC-3′) and a reverse primer that incorporated a 3′ NotI restriction site (5′-ATAAGAATGCGGCCGCTCAATTCTGGGCTTCTGGG-3′). The amplified sequence was then used to replace the EGFP open reading frame in pCMV-GFP with standard digestion and ligation to generate pCMV-Lin28a. pCMV-GFP was a gift from Dr. Connie Cepko (Addgene plasmid # 11153). pMSCV-mLin28A was a gift from Dr. George Daley (Addgene plasmid # 26357). The Lin28a-Flag open reading frame with a 5′ BamHI restriction site and a 3′ EcoRV restriction site was synthesized (codon optimized, gBlocks of Integrated DNA Technologies) and used to replace the EYFP open reading frame in pAAV-Ef1a-EYFP. pAAV-Ef1a-EYFP was a gift from Dr. Hongjun Song. pAAV-shPten was a gift from Dr. David Turner and Dr. Kevin Park. All restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs. Plasmids were amplified using DH5α competent cells (Thermo Fisher Scientific) and purified with Endofree plasmid maxi kit (QIAGEN).

Synaptotagmin chimeras were generated using site-directed mutagenesis. For chimeras Syt-1:7C2B₁, Syt-1:7C2B₂, and Syt-1:7C2B₃, wild-type Syt-1 pHluorin in the pCI vector was PCR amplified with primer sets designed to mutate each rat Syt-1 C2B loop to match the corresponding C2B loop of rat wild-type Syt-7 (Supplemental Table S1). The resulting amino acid substitutions for each construct are as follows: Syt-1:7C2B₁: K301A, V304I, L307T; Syt-1:7C2B₂: N333R, T334N; Syt-1:7C2B₃: Y364K, I367L, G368S, and K369R. Chimera Syt-1:7C2B₁₂₃ contains all of the above mutations and was generated by performing two additional rounds of mutagenesis; first, Syt-1:7C2B₁ was used as a template to mutate C2B loop 2, and then this product (Syt-1:7C2B₁₂) was used as the template in a second reaction to mutate C2B loop 3. Following PCR, all products were treated with DpnI restriction endonuclease (New England Biolabs, Ipswich, MA) to digest the template DNA, ligated, and transformed into DH5α competent cells (Thermo Fisher Scientific, Waltham, MA). Proper mutagenesis was confirmed using two subsequent rounds of Sanger sequencing (Applied Genomics Technology Center, Detroit, MI).

All plasmids were propagated in DH5α competent cells (Thermo Fisher Scientific). The plasmid DNA concentration was determined using a NanoDrop 2000 instrument (Thermo Fisher Scientific) by measuring the absorbance at 260 nm.

Total RNA was extracted from Hela cells using total RNA extraction kit, which was reversely transcribed into cDNA using reverse transcription kit (Solarbio, Beijing, China). tLyp-1 +  lamp2b gene was amplified by PCR using cDNA as template, purified and recovered by using gel recovery kit (Solarbio, Beijing, China). The pEGFP-C1 and tLyp-1-lamp2b genes were digested by Bgl II and BamH I enzymes, then connected by T4 ligase. The connection products were transformed into DH5α competent cells (stored at −80 °C, Thermo Scientific, Waltham, USA) and cultured for 12 h. Single colony was culled for plasmid extraction. Lipofectamine 3000 transfection reagent (Invitrogen, USA) were used to transfect pEGFP-C1 plasmid vector expressing tLyp-1-lamp2b fusion proteins into HEK293T cells.