Flag stx17

Manufactured by Addgene

FLAG-STX17 is a plasmid that encodes the human STX17 (Syntaxin 17) gene with an N-terminal FLAG tag. STX17 is a SNARE protein involved in autophagosome formation and trafficking. This plasmid can be used for the expression and purification of the FLAG-tagged STX17 protein in various cell lines.

Automatically generated - may contain errors

Lab products found in correlation

Gfp stx17, by Addgene (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions) Pcdna3 egfp, by Addgene (1 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Pt flag, by Addgene (1 mentions) Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions) Pmrfp lc3, by Addgene (1 mentions) Pmcherry c1, by Takara Bio (1 mentions) Pegfp c1, by Takara Bio (1 mentions) Pegfp n1, by Takara Bio (1 mentions)

4 protocols using flag stx17

Molecular Tagging of Autophagy Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Flag-Fis1 was kindly provided by Dr. Victor Chun-Kong Yu (National University of Singapore, Singapore) and then subcloned into mCherry-pXJ40 and GFP-pXJ40 vectors. Flag-STX17 (#45911) was purchased from Addgene and subcloned into pXJ40 vectors with Myc, GFP, mCherry, and BFP tags. GFP-Parkin was kindly provided by Dr. Kah-Leong Lim (National University of Singapore, Singapore)^{65 (link)}. Truncations of Fis1 and STX17 and site-directed mutations of STX17 were generated using a QuikChange II site-directed mutagenesis kit (Agilent Technologies). Myc-ULK1 and Myc-ATG5 were kindly provided by Dr. Shengcai Lin (Xiamen University, China). pEGFP-ATG14 (#21635), SECFP-ATG16L (#58994), GFP-WIPI-1 (#38272), GFP-DFCP1 (#86746), and retrovirus plasmid GFP-STX17 (#45909) were purchased from Addgene. GFP-Rab7, GFP-Rab5, and their site-directed mutants were kind gifts from Dr. Alexander Bershadsky (National University of Singapore, Singapore). Beclin1 was purchased from Addgene (#21150) and subcloned into Flag-pXJ40 vector. Mito-RFP and PSICOR-mt-Keima plasmids were kindly provided by Dr. Quan Chen (Nankai University, China). All primer sequences used for cloning are listed in Supplementary Data 1.

Xian H., Yang Q., Xiao L., Shen H.M, & Liou Y.C. (2019). STX17 dynamically regulated by Fis1 induces mitophagy via hierarchical macroautophagic mechanism. Nature Communications, 10, 2059.

+ Open protocol

+ Expand

Transfection and Serum Starvation of Fibroblasts

Check if the same lab product or an alternative is used in the 5 most similar protocols

The plasmids used for the transfection were obtained from Addgene: pt-FLAG (#31385), FLAG-STX17 (#45911), pcDNA3-EGFP (#13031), pEGFP-VAMP8 (#42311). CTL and DS fibroblasts were transfected with Lipofectamine3000 transfection reagent (L3000015) obtained from ThermoFisher Scientific. CTL (AG004392) and DS1 (AG006872) fibroblasts were plated on glass coverslips in 12-well plates with 1 ml of medium per well and left to reach 70% confluency. For each transfection, 2.5 μl of Lipofectamine3000, 1.5 μg of plasmid DNA and 1.5μl of P3000 reagent were diluted in 120 μl of OPTI-MEM reduced serum medium obtained from ThermoFisher Scientific (31985062) and incubated for 15 minutes at room temperature before addition to each well. The medium was replaced with fresh medium the next day and then the cells were used in IF experiments (NT = not treated or SS = 8h serum starvation).

Aivazidis S., Jain A., Rauniyar A.K., Anderson C.C., Marentette J.O., Orlicky D.J., Fritz K.S., Harris P.S., Siegel D., Maclean K.N, & Roede J.R. (2019). SNARE proteins rescue impaired autophagic flux in Down syndrome. PLoS ONE, 14(11), e0223254.

+ Open protocol

+ Expand

Plasmid Constructs for Autophagy Studies

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Plasmids used in the study were as follows: ptfLC3 (mRFP-GFP-LC3; Addgene plasmid #21074) and pmRFP-LC3 (Addgene plasmid #21075) were gifts from Tamotsu Yoshimori (Osaka University). FLAG-Stx17 (Addgene plasmid #45911) and FLAG-SNAP29 (Addgene plasmid#45915) were gifts from Noburu Mizushima (The University of Tokyo). GFP-VAMP8 was a gift from Thierry Galli (Institute of Psychiatry and Neuroscience of Paris [IPNP]) (Addgene plasmid #42311; Paumet et al., 2000 (link)), mCherry-DFCP1 was a gift from Do-Hyung Kim (University of Minnesota) (Addgene plasmid #86746; Kim et al., 2015 (link)), and HA-hATG14 was a gift from Noburu Mizushima (Addgene plasmid #24294; Itakura et al., 2008 (link)). Plasmid-containing HA-VPS33A was a kind gift from Mahak Sharma, IISER Mohali. Myc-Stx17 plasmid was a kind gift from Viktor Korolchuk, Newcastle University. GFP-LC3 plasmid was generated in the lab by excising out mRFP fragment from mRFP-GFP-LC3 plasmid.

Vats S, & Manjithaya R. (2019). A reversible autophagy inhibitor blocks autophagosome–lysosome fusion by preventing Stx17 loading onto autophagosomes. Molecular Biology of the Cell, 30(17), 2283-2295.

+ Open protocol

+ Expand

Plasmid Collection for Autophagy Research

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Plasmids encoding HTT103Q-GFP (1385; from Michael Sherman), FLAG-STX17 (45911; from Noboru Mizushima,) and GFP-STX17 (45909; from Noboru Mizushima,) were obtained from Addgene. Plasmids encoding MYC-BORCS6 and LAMP1-GFP were previously described.²¹ (link) A plasmid encoding GFP-mCherry-LC3B was generated by subcloning a mCherry-LC3B fragment into pEGFP-C1 (Clontech Laboratories, 6084). Plasmids encoding mCherry-VPS41 and mCherry-VPS39 were generated by subcloning the coding sequences from pSX-VPS41 and pSX-VPS39⁴⁹ (link) into pmCherry-C1 (Clontech Laboratories, 632524). A plasmid encoding KXD1-GFP was generated by subcloning a KXD1 fragment from MYC-KXD1²¹ (link) into pEGFP-N1 (Clontech Laboratories, 6085). The LIR-mutated KXD1 (KXD1(ΔLIR)-GFP) was generated by site-directed mutagenesis. The ARL8B-GFP plasmid was a gift from Dr. John Brumell (Hospital for Sick Children, Toronto, Canada). A FLAG-VPS41 plasmid was a gift from Dr. Chihiro Akazawa (Tokyo Medical and Dental University, Tokyo, Japan).

Jia R., Guardia C.M., Pu J., Chen Y, & Bonifacino J.S. (2017). BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy, 13(10), 1648-1663.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!