Memerald sec61β

Manufactured by Addgene

MEmerald-Sec61β is a fluorescent protein that can be used to visualize the endoplasmic reticulum (ER) in live cells. It consists of the Sec61β subunit fused to the mEmerald fluorescent protein. Sec61β is a component of the Sec61 translocon complex, which is responsible for the translocation of proteins into the ER.

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Lab products found in correlation

Mcherry sec61β, by Addgene (2 mentions) Hcas9, by Addgene (1 mentions) Pspcas9 bb 2a puro px459 v2, by Addgene (1 mentions) Aavs1 puro pgk1 3xflag twin strep, by Addgene (1 mentions) Pfuultra 2 fusion hotstart dna polymerase, by Agilent Technologies (1 mentions) Pmscarlet i c1, by Addgene (1 mentions) Gblock, by Integrated DNA Technologies (1 mentions) Pdsred2 er vector, by Takara Bio (1 mentions)

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MEmerald

4 protocols using memerald sec61β

LDAF1-FLAG-Seipin(1-310) Complex Expression

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The following plasmids were kind gifts: ERoxBFP (Addgene plasmid #68126) from Erik Snapp, mEmerald-Sec61β (Addgene plasmid #54249) from Michael Davidson, hCas9 (Addgene plasmid #41815) and gRNA-AAVS1-T2 (Addgene plasmid #41818) from George Church, AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid #68375) from Yannick Doyon, pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988) from Feng Zhang, and pmScarlet-i_C1 (Addgene plasmids #85044) from Dorus Gadella. pEGFP-N1 and pEGFP-C1 plasmids were purchased from Clontech Laboratories, pSMART-HC-Amp plasmid was purchased from Lucigen. pCAG-LNK vector was modified from pCAGEN (Addgene plasmid #11160) as described (Scheich et al., 2007).
For plasmid construction, all PCRs were performed using PfuUltra II Fusion HotStart DNA Polymerase (#600672, Agilent Technologies) and restriction enzymes were from New England Biolabs. The synthetic DNAs (gBlock, Integrated DNA Technologies) that were used in this study and cloning strategies of the other plasmids (including primer information) were summarized in Table S3 and S4, respectively.
For expression of the LDAF1-FLAG-seipin(1-310) complex, pCAG-LDAF1-FLAG-Seipin(1-310) plasmid was generated by pCAG-LNK-LDAF1-FLAG and pCAG-LNK-Seipin(1-310) using approach described in Scheich et al.(Scheich et al., 2007). The detailed cloning strategies were summarized in Table S3 and S4.

Chung J., Wu X., Lambert T.J., Lai Z.W., Walther T.C, & Farese RV J.r. (2019). Lipid droplet assembly factor-1 and seipin form a lipid droplet assembly complex. Developmental cell, 51(5), 551-563.e7.

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Cloning GFP-KDEL, SNAP-Sec61β, and Halo-Sec61β

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GFP-KDEL was cloned as previously described (Merta et al., 2021 (link)). SNAP-Sec61β and Halo-Sec61β were cloned as previously described (Bottanelli et al., 2016 (link)). mEmerald-Sec61β and mCherry-Sec61β were acquired from Addgene (plasmids 54249 and 49155, respectively).

Fuentes L.A., Marin Z., Tyson J., Baddeley D, & Bewersdorf J. (2023). The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ. bioRxiv.

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Engineered Fluorescent Sec61β Constructs

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mEmerald-Sec61β and mCherry-Sec61β were acquired from Addgene (plasmids 54249 and 49155, respectively). GFP-KDEL was cloned as previously described (Merta et al., 2021 (link)). In brief, a pDsRed2-ER vector (632409; Clontech) was digested with AgeI and HindIII to remove DsRed2, and a PCR-amplified GFP sequence with AgeI and HindIII sites was ligated into the vector. SNAP-Sec61β and Halo-Sec61β were cloned as previously described (Bottanelli et al., 2016 (link)). Briefly, mEmerald-Sec61β (above) was digested with NheI and BgIII to remove mEmerald, and a PCR-amplified HaloTag or SNAP-tag sequence with NheI and BgIII sites was ligated in mEmerald’s place.

Fuentes L.A., Marin Z., Tyson J., Baddeley D, & Bewersdorf J. (2023). The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ. The Journal of Cell Biology, 222(10), e202301112.

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Mitochondrial Fluorescent Protein Constructs

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Mito-DsRed plasmid, a gift from Dr. Quan Chen (Institute of Zoology, CAS), was constructed by fusing the mitochondrial target sequence of human TXN2 with the DsRed sequence. TOM20-GFP and mito-YFP, gifts from Dr. Li Yu (Tsinghua University)^{11 (link)}, were obtained by inserting the rat TOM20 coding sequence into pEGFP-N1 and by fusing the mitochondrial target sequence of human COX8A with the YFP sequence, respectively. We constructed the mito-BFP plasmid by replacing the DsRed of mito-DsRed with the BFP sequence. We also constructed TFAM-GFP by inserting the human TFAM coding sequence into pEGFP-N1 and TFAM-mCherry by replacing the GFP of TFAM-GFP with the mCherry sequence. TFAM-HaloTag was a gift from Dr. Dong Li (Institute of Biophysics, CAS) and mCherry-KDEL was a gift from Dr. Jianguo Chen (Peking University). We purchased mEmerald-Sec61β from Addgene (#90992). The primers used in this study are listed in Supplementary Table 1.

Qin J., Guo Y., Xue B., Shi P., Chen Y., Su Q.P., Hao H., Zhao S., Wu C., Yu L., Li D, & Sun Y. (2020). ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation. Nature Communications, 11, 4471.

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