Mouse KCNH6 and Munc-18-1 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy and were verified by DNA sequencing. The sequences of the primers used are listed in Supplementary Table 2. These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG vector (Invitrogen) or mCherry-C1 vector (Clontech) as described previously [24 (link)]. Insulin-EGFP was generated as described previously [25 (link)]. To generate recombinant adenoviruses, KCNH6 WT and KCNH6 R246A/T248A/L250A (3A) mutant were inserted into pENTR-3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen), which co-produces red fluorescent protein (Cherry) to allow identification of transfected cells. To express an exogenous protein, HEK293A cells were transfected with the plasmids using Lipofectamine 3000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses.
Mcherry c1 vector
Manufactured by Takara Bio
The MCherry-C1 vector is a plasmid designed for the expression of proteins fused to the mCherry fluorescent protein in mammalian cells. mCherry is a red fluorescent protein derived from the Discosoma species of coral. The vector allows for the insertion of the gene of interest upstream of the mCherry coding sequence, enabling the expression of the target protein as a fusion with mCherry.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3 flag vector, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3 ha, by Thermo Fisher Scientific (1 mentions)
Pentr3c, by Thermo Fisher Scientific (1 mentions)
Lr recombination clonase, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
5 protocols using mcherry c1 vector
1
Adenoviral Expression of KCNH6 Mutants
2
Cloning CHMP6 into mCherry-C1 Vector
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The CHMP6 sequence was amplified by PCR from Flag-CHMP6 plasmid and cloned to mCherry-C1 vector (Clontech).
3
Generation of HDAC9 Deletion Construct
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The pCAG-EYFP, pCAGGS-HDAC9-EGFP and pCAGGS-HDAC9-S218/448A-EGFP constructs were previously described9 (link), 26 (link). To generate a vector expressing HDAC9 lacking the MEF2-binding domain, a deletion corresponding to amino acid residues 135–152 of HDAC9 cDNA was introduced into pCAGGS-HDAC9-S218/448A-EGFP by PCR using the mutagenic primer 5′-AGCTTCCTCCTCTCAGAGGCAAAGATAGAGGACGAAGTAAATCAGCAACAAAAGACACTCCAA-3′ and its antisense strand25 (link).
The pCAG-mCherry plasmid was constructed by PCR amplification of the mCherry ORF from mCherry-C1 vector (Clontech) and ligation into a pGEM-T Easy Vector (Promega, Madison, WI), followed by subcloning of the ORF into the EcoRI site of the pCAGGS vector.
The pCAG-mCherry plasmid was constructed by PCR amplification of the mCherry ORF from mCherry-C1 vector (Clontech) and ligation into a pGEM-T Easy Vector (Promega, Madison, WI), followed by subcloning of the ORF into the EcoRI site of the pCAGGS vector.
4
Construction and Validation of Fluorescent Protein Fusion Constructs
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The PMP34-GFP66 (link) and PMP34-GFP-UBko19 (link) constructs used in this study were previously generated using standard protocols, where the PCR product of the ORF of PMP34 was ligated into the EcoRI and BamHI site of the pmGFP-N1 vector (Clontech). Ub-KO (G76V), a ubiquitin where all seven lysines were mutated to arginines, was excised from GFP-Ub-KO (G76V) (Addgene 11932) with BsrGI and NotI, and the resulting fragments were ligated into similar sites in PMP34-GFP. The Cherry-LC3 construct was previously generated27 , where the PCR product of the LC3-B ORF was ligated into the BglII-EcoRI site of the mCherry-C1 vector (Clontech) using standard protocols. LAMP1A-mEmerald was donated by Dr. Sergio Grinstein (Hospital for Sick Children, Toronto, ON, Canada), and FLAG-ULK1-wild type was donated by Dr. John Brumell (Hospital for Sick Children, Toronto, ON, Canada). The siRNAs used in this study were custom synthesized from Sigma-Aldrich and are listed in Supplementary Table 2 . siRNA knockdown was validated by immunoblot or TaqMan real-time quantitative PCR.
5
Zebrafish ESCRT Protein Expression
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Cep55l, Tsg101, Chmp4bb, Chmp4ba zebrafish (Danio rerio) coding sequences were amplified by PCR from zebrafish cDNA using specific primers (Table S1) and subcloned into mCherry-C1 vector (Clontech, Mountain View, CA). hVPS4A in pEGFP-C1 was previously described in Elia et. al. (33) . All plasmids then subcloned into pCS2+ vector (kindly provided by Gil Levkowitz, Weizmann Institute of Science, Israel). mRNA encoding for mCherry-cep55 mCherry-tsg101, mCherry-chmp4bb, mCherry-chmp4ba were generated by in-vitro transcribed using mMessage mMachine SP6 kit (AM-1340 Invitrogen) and purified using RNeasy mini kit (74104 Qiagen). Morpholino oligos against chmp4bb and standard control oligos (Gene Tools, Inc.) were designed and managed according to manufacture protocol (Table S1). Two morpholino oligos were injected and analyzed by western blot analysis for chmp4bb knock down (MO_1chmp4bb and MO_2chmp4bb) (Fig. S4D). MO_1chmp4bb was found to be more efficient by western blot analysis, and was therefore used in all the presented experiments.
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