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Addgene inc pmxs ip gfp ulk1
GABARAPs do not translocate to damaged mitochondria and early stages of autophagosome biogenesis mediated by WIPI1 and DFCP1 are inhibited in autophagy receptor deficient cell lines Representative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCherry-Parkin (mCh-Parkin) and either ( a ) <t>GFP-tagged</t> GABARAP, GABARAPL1 or GABARAPL2, ( b ) GFP-WIPI1 or ( c ) GFP-DFCP1 immunostained for Tom20 (n=3 for each condition, see Figure 4b, c for quantification of b and c ). d, mCh-Parkin cell lines as indicated were subjected to either Phos-Tag SDS-PAGE or standard SDS-PAGE followed by immunoblotting. Arrows indicate the position of phosphorylated Beclin species. e, Representative images of untreated WT, N/O (NDP52/OPTN) DKO and pentaKO cell lines expressing mCh-Parkin and <t>GFP-ULK1</t> were immunostained for Tom20 and GFP (n=3). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.
Pmxs Ip Gfp Ulk1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy"

Article Title: The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy

Journal: Nature

doi: 10.1038/nature14893

GABARAPs do not translocate to damaged mitochondria and early stages of autophagosome biogenesis mediated by WIPI1 and DFCP1 are inhibited in autophagy receptor deficient cell lines Representative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCherry-Parkin (mCh-Parkin) and either ( a ) GFP-tagged GABARAP, GABARAPL1 or GABARAPL2, ( b ) GFP-WIPI1 or ( c ) GFP-DFCP1 immunostained for Tom20 (n=3 for each condition, see Figure 4b, c for quantification of b and c ). d, mCh-Parkin cell lines as indicated were subjected to either Phos-Tag SDS-PAGE or standard SDS-PAGE followed by immunoblotting. Arrows indicate the position of phosphorylated Beclin species. e, Representative images of untreated WT, N/O (NDP52/OPTN) DKO and pentaKO cell lines expressing mCh-Parkin and GFP-ULK1 were immunostained for Tom20 and GFP (n=3). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.
Figure Legend Snippet: GABARAPs do not translocate to damaged mitochondria and early stages of autophagosome biogenesis mediated by WIPI1 and DFCP1 are inhibited in autophagy receptor deficient cell lines Representative images of WT, N/O (NDP52/OPTN) DKO and pentaKOs expressing mCherry-Parkin (mCh-Parkin) and either ( a ) GFP-tagged GABARAP, GABARAPL1 or GABARAPL2, ( b ) GFP-WIPI1 or ( c ) GFP-DFCP1 immunostained for Tom20 (n=3 for each condition, see Figure 4b, c for quantification of b and c ). d, mCh-Parkin cell lines as indicated were subjected to either Phos-Tag SDS-PAGE or standard SDS-PAGE followed by immunoblotting. Arrows indicate the position of phosphorylated Beclin species. e, Representative images of untreated WT, N/O (NDP52/OPTN) DKO and pentaKO cell lines expressing mCh-Parkin and GFP-ULK1 were immunostained for Tom20 and GFP (n=3). OA, Oligomycin and Antimycin A. Scale bars, 10 μm.

Techniques Used: Expressing, SDS Page

Characterization of autophagy receptor function during mitophagy mCherry-Parkin (mCh-Parkin) expressing WT, N/O (NDP52/OPTN) DKO and pentaKOs were quantified for a, GFP-LC3A, LC3B and LC3C translocation to mitochondria, b, GFP-WIPI1 or c, GFP-DFCP1 structures per cell ( > 100 cells counted for each sample) or d, were immunoblotted using phospho-specific anti-S757 and S317 ULK1 antibodies. e, mCherry-Parkin WT, N/O DKO and pentaKOs stably expressing GFP-ULK1 were quantified for GFP-ULK1 puncta per cell (left graph) and the percentage of those puncta on mitochondria (right graph). f, Representative data of e , cells were immunostained for Tom20 and GFP. g, pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCherry-ULK1 (mCh-ULK1) and myc-tagged receptors, were treated with rapalog then imaged live. h, Quantification of mitochondrial ULK1 puncta in g . i , Quantification of mitochondrial ULK1 puncta in pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCh-ULK1 and myc-OPTN mutants, treated with rapalog then imaged live. Quantification in a , b, c, e, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA. (*** P
Figure Legend Snippet: Characterization of autophagy receptor function during mitophagy mCherry-Parkin (mCh-Parkin) expressing WT, N/O (NDP52/OPTN) DKO and pentaKOs were quantified for a, GFP-LC3A, LC3B and LC3C translocation to mitochondria, b, GFP-WIPI1 or c, GFP-DFCP1 structures per cell ( > 100 cells counted for each sample) or d, were immunoblotted using phospho-specific anti-S757 and S317 ULK1 antibodies. e, mCherry-Parkin WT, N/O DKO and pentaKOs stably expressing GFP-ULK1 were quantified for GFP-ULK1 puncta per cell (left graph) and the percentage of those puncta on mitochondria (right graph). f, Representative data of e , cells were immunostained for Tom20 and GFP. g, pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCherry-ULK1 (mCh-ULK1) and myc-tagged receptors, were treated with rapalog then imaged live. h, Quantification of mitochondrial ULK1 puncta in g . i , Quantification of mitochondrial ULK1 puncta in pentaKOs expressing FRB-Fis1, PINK1Δ110-YFP-2xFKBP, mCh-ULK1 and myc-OPTN mutants, treated with rapalog then imaged live. Quantification in a , b, c, e, h and i are mean ± s.d. from 3 independent experiments and use one-way ANOVA. (*** P

Techniques Used: Expressing, Translocation Assay, Stable Transfection

OPTN and NDP52 rescue DFCP1 and ULK1 recruitment deficit in pentaKOs a, Representative images of pentaKOs expressing mCherry-Parkin (mCh-Parkin), GFP-DFCP1 and the indicated FLAG/HA-tagged autophagy receptors immunostained for HA (n=2). Right-hand panels display co-localization of FLAG/HA-tagged constructs and GFP-DFCP1 by fluorescence intensity line measurement. b, Representative images of pentaKOs expressing mCherry-Parkin and GFP-ULK1 were rescued with FLAG/HA-OPTN, FLAG/HA-NDP52, and FLAG/HA-p62, and immunostained for HA and GFP. Arrows indicate HA-tagged receptor puncta (n=2). Right panels display colocalization of HA and GFP by fluorescence intensity line measurement. c, d, Representative images of pentaKOs stably expressing FRB-Fis1 and transiently expressing PINK1Δ110-YFP-2xFKBP and vector or myc-tagged receptors, were ( c ) untreated or ( d ) treated with rapalog and imaged live (n=3, see Figure 4h, i for quantification of c , d ). OA, Oligomycin and Antimycin A. Scale bars, 10 μm. e, Old and new models of PINK1/Parkin mitophagy. The old model is dominated by Parkin ubiquitination of mitochondrial proteins. Here PINK1 plays a small initiator role whose main function is to bring Parkin to the mitochondria. The new model depicts Parkin-dependent and independent pathways leading to robust and low-level mitophagy, respectively. Based on our data, PINK1 is central to mitophagy both before and after Parkin recruitment by phosphorylating UB to recruit both Parkin and autophagy receptors mitochondria, to induce clearance. In the absence of Parkin (right panel), this occurs at a low level due to the relatively low basal UB on mitochondria. When Parkin is present it serves to amplify the PINK1 generated UB-PO 4 signal, allowing for robust and rapid mitophagy induction.
Figure Legend Snippet: OPTN and NDP52 rescue DFCP1 and ULK1 recruitment deficit in pentaKOs a, Representative images of pentaKOs expressing mCherry-Parkin (mCh-Parkin), GFP-DFCP1 and the indicated FLAG/HA-tagged autophagy receptors immunostained for HA (n=2). Right-hand panels display co-localization of FLAG/HA-tagged constructs and GFP-DFCP1 by fluorescence intensity line measurement. b, Representative images of pentaKOs expressing mCherry-Parkin and GFP-ULK1 were rescued with FLAG/HA-OPTN, FLAG/HA-NDP52, and FLAG/HA-p62, and immunostained for HA and GFP. Arrows indicate HA-tagged receptor puncta (n=2). Right panels display colocalization of HA and GFP by fluorescence intensity line measurement. c, d, Representative images of pentaKOs stably expressing FRB-Fis1 and transiently expressing PINK1Δ110-YFP-2xFKBP and vector or myc-tagged receptors, were ( c ) untreated or ( d ) treated with rapalog and imaged live (n=3, see Figure 4h, i for quantification of c , d ). OA, Oligomycin and Antimycin A. Scale bars, 10 μm. e, Old and new models of PINK1/Parkin mitophagy. The old model is dominated by Parkin ubiquitination of mitochondrial proteins. Here PINK1 plays a small initiator role whose main function is to bring Parkin to the mitochondria. The new model depicts Parkin-dependent and independent pathways leading to robust and low-level mitophagy, respectively. Based on our data, PINK1 is central to mitophagy both before and after Parkin recruitment by phosphorylating UB to recruit both Parkin and autophagy receptors mitochondria, to induce clearance. In the absence of Parkin (right panel), this occurs at a low level due to the relatively low basal UB on mitochondria. When Parkin is present it serves to amplify the PINK1 generated UB-PO 4 signal, allowing for robust and rapid mitophagy induction.

Techniques Used: Expressing, Construct, Fluorescence, Stable Transfection, Plasmid Preparation, Generated

Related Articles

Clone Assay:

Article Title: The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens
Article Snippet: .. Human HMGB1 cloned into plasmid mEGF-C1 to produce GFP-HMGB1 fusion proteins was also prepared. mCherry2-C1 and mEGFP-C1 were gifts from Michael Davidson (Addgene plasmids 54563 and 54759). .. BALB/c mice purchased from The Jackson Laboratory (Bar Harbor, ME) were housed at the OSU animal facility.

Article Title: The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy
Article Snippet: .. Cloning and generation of stable cell lines pMXs-puro-GFP-WIPI1 and pMXs-puro-GFP-DFCP1 were a kind gift from Dr. N. Mizushima (University of Toyko, Japan) and pMXs-IP-GFP-ULK1 was purchased from Addgene (#38193). .. To generate pBMN-mEGFP-C1, mEGFP-C1 (Addgene #36412) was PCR amplified (together with the multiple cloning site) and cloned into pBMN-Z at BamHI/SalI sites using the Gibson Cloning kit (New England BioLabs) according to manufacturer's instructions.

Transfection:

Article Title: Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis
Article Snippet: .. For stable transfection with LAMP1-mCherry, 24 hr after transfection, cells were subjected to G-418 selection (400 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) in the growth medium. pLAMP1-mCherry was a gift from Amy Palmer (Addgene plasmid # 45147). pCMV-lyso-pHluorin was a gift from Christian Rosenmund (Addgene plasmid # 70113). pTurbo-RFP-C was from Evrogen (Moscow, Russia). .. Live cell imaging HeLa cells were plated in 24-well glass bottom plates (In Vitro Scientific, CA, USA).

Selection:

Article Title: Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis
Article Snippet: .. For stable transfection with LAMP1-mCherry, 24 hr after transfection, cells were subjected to G-418 selection (400 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) in the growth medium. pLAMP1-mCherry was a gift from Amy Palmer (Addgene plasmid # 45147). pCMV-lyso-pHluorin was a gift from Christian Rosenmund (Addgene plasmid # 70113). pTurbo-RFP-C was from Evrogen (Moscow, Russia). .. Live cell imaging HeLa cells were plated in 24-well glass bottom plates (In Vitro Scientific, CA, USA).

Stable Transfection:

Article Title: Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis
Article Snippet: .. For stable transfection with LAMP1-mCherry, 24 hr after transfection, cells were subjected to G-418 selection (400 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) in the growth medium. pLAMP1-mCherry was a gift from Amy Palmer (Addgene plasmid # 45147). pCMV-lyso-pHluorin was a gift from Christian Rosenmund (Addgene plasmid # 70113). pTurbo-RFP-C was from Evrogen (Moscow, Russia). .. Live cell imaging HeLa cells were plated in 24-well glass bottom plates (In Vitro Scientific, CA, USA).

Article Title: The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy
Article Snippet: .. Cloning and generation of stable cell lines pMXs-puro-GFP-WIPI1 and pMXs-puro-GFP-DFCP1 were a kind gift from Dr. N. Mizushima (University of Toyko, Japan) and pMXs-IP-GFP-ULK1 was purchased from Addgene (#38193). .. To generate pBMN-mEGFP-C1, mEGFP-C1 (Addgene #36412) was PCR amplified (together with the multiple cloning site) and cloned into pBMN-Z at BamHI/SalI sites using the Gibson Cloning kit (New England BioLabs) according to manufacturer's instructions.

Over Expression:

Article Title: Subcellular Localization of Sprouty2 in Human Glioma Cells
Article Snippet: .. For transient overexpression of Sprouty2, pmNeonGreen-C1-hSprouty2 plasmid was used (mNeonGreen was licensed from Allele Biotech; Shaner et al., ). mCherry-Rab5a was a gift from Michael Davidson (Addgene #55126), pLAMP1-mCherry a gift from Amy Palmer (Addgene #45147) and C1-mCherry-Rab11a a gift from Lukas Huber (Biocenter Innsbruck). .. For shRNA-mediated Sprouty2 depletion, annealed oligonucleotide targeting Sprouty2 (shRNA Target1: GCAGGTACATGTCTTGTCT, shRNA Target2: GATCAGAGCCATCCGAAAC) was inserted into pGLTR-puro plasmid for a stable and conditional RNAi system.

Plasmid Preparation:

Article Title: The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens
Article Snippet: .. Human HMGB1 cloned into plasmid mEGF-C1 to produce GFP-HMGB1 fusion proteins was also prepared. mCherry2-C1 and mEGFP-C1 were gifts from Michael Davidson (Addgene plasmids 54563 and 54759). .. BALB/c mice purchased from The Jackson Laboratory (Bar Harbor, ME) were housed at the OSU animal facility.

Article Title: Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching
Article Snippet: .. This plasmid was prepared by Gibson assembly employing sequences from commercial plasmids pmTurquoise2-ER (Addgene #36204), from which the insert was taken, and pLAMP1-mCherry (Addgene 45147), which served as backbone, employing the following primers: insert forward (5′-TATGGTGAGCAAGGGCGAGGAG-3′), insert reverse (5′-ACAGCTCGTCCATGCCGAGAGT-3′), backbone forward (5′-GGCATGGACGAGCTGTACAAGGCTAC-3′) and backbone reverse (5′-TCGCCCTTGCTCACCATACCG-3′). .. The insert and backbone DNA fragments were generated by polymerase chain reaction (PCR) using Phusion High-Fidelity PCR Master Mix with HF buffer (NEB, M0531S).

Article Title: Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis
Article Snippet: .. For stable transfection with LAMP1-mCherry, 24 hr after transfection, cells were subjected to G-418 selection (400 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) in the growth medium. pLAMP1-mCherry was a gift from Amy Palmer (Addgene plasmid # 45147). pCMV-lyso-pHluorin was a gift from Christian Rosenmund (Addgene plasmid # 70113). pTurbo-RFP-C was from Evrogen (Moscow, Russia). .. Live cell imaging HeLa cells were plated in 24-well glass bottom plates (In Vitro Scientific, CA, USA).

Article Title: Subcellular Localization of Sprouty2 in Human Glioma Cells
Article Snippet: .. For transient overexpression of Sprouty2, pmNeonGreen-C1-hSprouty2 plasmid was used (mNeonGreen was licensed from Allele Biotech; Shaner et al., ). mCherry-Rab5a was a gift from Michael Davidson (Addgene #55126), pLAMP1-mCherry a gift from Amy Palmer (Addgene #45147) and C1-mCherry-Rab11a a gift from Lukas Huber (Biocenter Innsbruck). .. For shRNA-mediated Sprouty2 depletion, annealed oligonucleotide targeting Sprouty2 (shRNA Target1: GCAGGTACATGTCTTGTCT, shRNA Target2: GATCAGAGCCATCCGAAAC) was inserted into pGLTR-puro plasmid for a stable and conditional RNAi system.

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