ptripz addgene  (Addgene inc)

Journal: eLife

Article Title: Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

doi: 10.7554/elife.79116

Figure Lengend Snippet: Figure 1. Detection of the subcellular localization of GDOWN1 or BiFC signal between GDOWN1 and some transcription related factors. (A) The ectopically expressed human GDOWN1 in HeLa cells was stringently localized in the cytoplasm. Human GDOWN1 proteins fused with indicated tags, including a fluorescent tag at either terminus, a Flag tag alone or together with two NLS motifs, were ectopically expressed in HeLa cells and the subcellular localization was detected by directly monitoring the fluorescent signal or by immunofluorescence assays (IF) using an anti-FLAG antibody. (B) The endogenous human or mouse GDOWN1 was stringently located in the cytoplasm. Each indicated cell line was fractionated to separate the cytosol from the nuclei, and the cytoplasmic fraction, C,the nuclear fraction, N, and the whole cell lysate, T (total), were further detected by WB. α-TUBULIN and FBL (a nucleolus protein) were used as markers of the cytoplasmic and nuclear fractions, respectively. (C) GDOWN1 remained in the cytoplasm upon LMB treatment. The indicated cell lines were subjected to either mock or LMB treatment (detailed below) before further fractionation and WB analyses. (D) BiFC analyses of the protein-protein interactions between GDOWN1 and its potential binding partners. (E) BiFC analyses of the protein-protein interactions between NEFL-E•NEFL-A, SPT4•SPT5, GDOWN1•GDOWN1 or 3xNLS-GDOWN1•3xNLS-GDOWN1. Proteins of interest were cloned and fused with either VN (the N-terminus of Venus) or YC (the C-terminus of YFP), and each of the indicated pair of plasmids was co-transfected into HeLa cells, and the confocal microscopy images were acquired 24 hr post transfection. The LMB treatment was carried out at 20 nM final concentration for 6 hr and the mock treatment was done with an equal volume of ethanol in parallel. Nuclear DNA was stained by Hoechst 33342. scale bars—30 μm. Without further labeled with details, the GDOWN1 antibody used in WB assays was the one generated from rabbits.

Article Snippet: DOI: https://doi.org/10.7554/eLife.79116 20 of 32 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pBiFC (VN- or YC-) (plasmid) Provided by Dr. Tom Kerppola, University of Michigan Medical School, USA Plasmids used in BiFC assay Recombinant DNA reagent pTripZ (plasmid) Addgene #127696 Lentiviral vector for inducible expression in mammalian cells Recombinant DNA reagent pMD2.G (plasmid) Addgene #12259 Lentivirus packaging vector Recombinant DNA reagent psPAX2 (plasmid) Addgene #12260 Lentivirus packaging vector Recombinant DNA reagent pcDNA3.1(+) (plasmid) Addgene #78110 Gene expression vector Recombinant DNA reagent pX459 (plasmid) Addgene #118632 CRISPR- Cas9 vector Antibody anti- GDOWN1 (Rabbit polyclonal) In this study, generated in Biodragon, Suzhou, China Immunogen: human GDOWN1 (251– 368 aa); WB: 1:1000 Preferably used in this study without further indication.

Techniques: FLAG-tag, Immunofluorescence, Fractionation, Protein-Protein interactions, Binding Assay, Clone Assay, Transfection, Confocal Microscopy, Concentration Assay, Staining, Labeling, Generated

Journal: eLife

Article Title: Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

doi: 10.7554/elife.79116

Figure Lengend Snippet: Figure 2. Identification of the Nuclear Export Signal (NES) motifs in GDOWN1. (A) A diagram of human GDOWN1 and its mutants used in the IF or BiFC-based motif screening analyses. The mutants whose names are marked in red are the ones translocated into the nucleus in response to LMB treatment. The sequences of the identified NES motifs are shown in yellow circles and the positions are labled on each side, and the core amino acids selected for mutagenesis are highlighted in red. (B) Identification of the NES motifs in GDOWN1 via IF or BiFC-based screening analyses. Left panel:

Article Snippet: DOI: https://doi.org/10.7554/eLife.79116 20 of 32 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pBiFC (VN- or YC-) (plasmid) Provided by Dr. Tom Kerppola, University of Michigan Medical School, USA Plasmids used in BiFC assay Recombinant DNA reagent pTripZ (plasmid) Addgene #127696 Lentiviral vector for inducible expression in mammalian cells Recombinant DNA reagent pMD2.G (plasmid) Addgene #12259 Lentivirus packaging vector Recombinant DNA reagent psPAX2 (plasmid) Addgene #12260 Lentivirus packaging vector Recombinant DNA reagent pcDNA3.1(+) (plasmid) Addgene #78110 Gene expression vector Recombinant DNA reagent pX459 (plasmid) Addgene #118632 CRISPR- Cas9 vector Antibody anti- GDOWN1 (Rabbit polyclonal) In this study, generated in Biodragon, Suzhou, China Immunogen: human GDOWN1 (251– 368 aa); WB: 1:1000 Preferably used in this study without further indication.

Techniques: Mutagenesis

Journal: eLife

Article Title: Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

doi: 10.7554/elife.79116

Figure Lengend Snippet: Figure 3. Identification and mechanistic analyses of the Cytoplasmic Anchoring Signal (CAS) motif in GDOWN1. (A) A diagram of human GDOWN1 and its mutants used in the BiFC-based motif screening analyses. The mutants whose names are marked in red are the ones translocated into the nucleus in response to LMB treatment. The sequences of the identified NES or CAS motif are shown in yellow circles and the core amino acids selected for mutagenesis are highlighted in red. (B) Identification of the second NES and the CAS motif in GDOWN1 via BiFC-based screening analyses. The experiments were carried out in the same way as described in Figure 2B. (C) The enrichment of GDOWN1 at the nuclear pore region was regulated by the CAS motif. HeLa cells stably expressing the wild type GDOWN1 (WT-Venus) or the CAS mutant (mCAS-Venus) were used for detection. The nuclear membrane was approximately represented via IF using an antibody against the nuclear lamina (α-LAMIN-A/C). Confocal Images were collected and further zoomed in for 3 folds to show more details of the nuclear membranes. (D) BiFC analyses of the interactions between GDOWN1 and some subunits of NPC in HeLa cells. Upper panel: a simplified diagram of an NPC; lower panel: BiFC results between GDOWN1 (or its CAS mutant) and the indicated NPC components. (E) Detection of the interaction between GDOWN1 and NUP214 by IP-WB. Parental Hela cells or HeLa cells stably expressed GDOWN1(WT or mCAS)-Venus-Flag were employed in IP experiment using a Flag antibody and further detected by WB with indicated antibodies. The LMB treatment was carried out as previously described. The nuclear DNA was stained with Hoechst 33342. The scale bars represent 30 μm except for the ones in C represent 15 μm.

Article Snippet: DOI: https://doi.org/10.7554/eLife.79116 20 of 32 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pBiFC (VN- or YC-) (plasmid) Provided by Dr. Tom Kerppola, University of Michigan Medical School, USA Plasmids used in BiFC assay Recombinant DNA reagent pTripZ (plasmid) Addgene #127696 Lentiviral vector for inducible expression in mammalian cells Recombinant DNA reagent pMD2.G (plasmid) Addgene #12259 Lentivirus packaging vector Recombinant DNA reagent psPAX2 (plasmid) Addgene #12260 Lentivirus packaging vector Recombinant DNA reagent pcDNA3.1(+) (plasmid) Addgene #78110 Gene expression vector Recombinant DNA reagent pX459 (plasmid) Addgene #118632 CRISPR- Cas9 vector Antibody anti- GDOWN1 (Rabbit polyclonal) In this study, generated in Biodragon, Suzhou, China Immunogen: human GDOWN1 (251– 368 aa); WB: 1:1000 Preferably used in this study without further indication.

Techniques: Mutagenesis, Stable Transfection, Expressing, Membrane, Staining