Journal: eLife

Article Title: Controlling motor neurons of every muscle for fly proboscis reaching

doi: 10.7554/eLife.54978

Figure Lengend Snippet:

Article Snippet: Antibody , rat mAb anti-FLAG , Novus Biologicals , Cat# NBP1-06712; RRID: AB_1625981 , (1:200) , .

Techniques: Muscles, Construct, Software

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Role of the di-proline motif in degradation of Δ9-desaturase. N-terminal amino acid sequences of Δ9-desaturases were compared (D. melanogaster DESAT1 (NP_652731), Mus musculus SCD1 (NP_033153), M. musculus SCD3 (NP_077770), Homo sapiens SCD1 (NP_005054), Danio rerio stearoyl-CoA desaturase (AAO25582), and S. cerevisiae Ole1 (CAA96757)) (A). Schematic illustration of DESAT1, mouse SCD1, and constructed mutants is shown (B). S2 cells expressing mouse SCD1-FLAGC, chimera-FLAGC, chimera (AA)-FLAGC, and SCD1 (di-Pro)-FLAGC were treated with C18:1 (100 μm) for 6 h (C) or DESAT1 inhibitor 37c (1 μm) for 16 h (D), and the amounts of endogenous DESAT1, FLAG-tagged exogenous Δ9-desaturases, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively. Band intensities were determined by ImageJ software, and levels of SCD1 proteins are shown relative to the amount of SCD1 protein in vehicle-treated cells (C and D). Mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Construct, Expressing, Software

Journal: The Journal of Biological Chemistry

Article Title: An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

doi: 10.1074/jbc.M117.801936

Figure Lengend Snippet: Identification of protease involved in the degradation of DESAT1. S2 cells were treated with calpeptin (50 μm), MG132 (50 μg/ml), or chloroquine (50 μm) for 6 h (A). S2 cells were treated with dsRNA against GFP, CalpA, CalpB, CalpC, or sol for 72 h and then exposed to C18:1 (100 μm) for 6 h (B). S2 cells expressing DESAT1-FLAGC and DESAT1 (P2A/P3A)-FLAGC were treated with calpeptin (50 μm) for 6 h (C). The amounts of endogenous DESAT1, FLAG-tagged exogenously expressed DESAT1, and α-tubulin protein were detected with anti-DESAT1 antibody, anti-FLAG antibody, and anti-α-tubulin antibody, respectively (A–C). Band intensities were determined by ImageJ software, and levels of DESAT1 proteins are shown relative to the amount of DESAT1 protein in vehicle-treated cells (A–C). Mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: Anti-α-tubulin antibody (PM054), anti-FLAG antibody (M2), and anti-ubiquitin antibody (SPC-119) were purchased from MBL, Sigma, and StressMarq Biosciences, respectively.

Techniques: Expressing, Software

Journal: eLife

Article Title: Cell types and neuronal circuitry underlying female aggression in Drosophila

doi: 10.7554/eLife.58942

Figure Lengend Snippet:

Article Snippet: Antibody , Rat α-FLAG Tag (DYKDDDDK Epitope tag) monoclonal , Novus Biologicals , Cat #: NBP1-06712 , Dilution (1:200).

Techniques: Electron Microscopy

Journal:

Article Title: Patterns of Gene Expression and a Transactivation Function Exhibited by the vGCR (ORF74) Chemokine Receptor Protein of Kaposi's Sarcoma-Associated Herpesvirus

doi: 10.1128/JVI.76.7.3421-3439.2002

Figure Lengend Snippet: Detection of the KSHV vGCR protein by Western immunoblotting in transfected cells and after in vitro translation. Expression vector plasmid DNA was transfected into 293T cells, and protein extracts were fractionated by gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with either rabbit anti-vGCR-N antibody (lanes 1 to 4) or anti-Flag epitope antibody (lanes 5 to 8). Lanes 1 and 5, extract from cells receiving SV2-vCGR/Flag DNA; 2 and 6, SV2-ORF-K1A/Flag; 3 and 7, SV2-ORF-K1B/Flag; 4 and 8, untransfected 293T cell controls. Lane 9, autoradiograph of gel electrophoretically fractionated 35S-labeled protein products following in vitro transcription-translation from the SV2-vGCR expression plasmid (pGH398a). Positions of stained protein size markers are indicated (in kilodaltons).

Article Snippet: Other antibodies used included mouse M2 anti-Flag epitope monoclonal antibody (MAb) (Kodak), mouse MAb against the KSHV ORF59 DNA replication protein ( 17 ), mouse MAb LN53 against the LANA1 protein ( 39 ), rabbit antipeptide PAbs against KSHV vIL6 (K2) ( 10 ) and KSHV ZMP-A (K5) ( 9 ), rhodamine-labeled goat anti-mouse immunoglobulin (Ig) secondary MAb, and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit Ig secondary MAb (Chemicon or Jackson Immunoresearch).

Techniques: Western Blot, Transfection, In Vitro, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Incubation, FLAG-tag, Autoradiography, Labeling, Staining

Journal: PLoS ONE

Article Title: Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin

doi: 10.1371/journal.pone.0129965

Figure Lengend Snippet: A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by post-translational modifications are shown, S10 as the main O -GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf K13R , ΔLf K308R , ΔLf K361R , ΔLf K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ≥ 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ≥ 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ≥ 3).

Article Snippet: Antibodies against the 3xFLAG epitope (mouse monoclonal anti-FLAG M2 antibody, Sigma), HA epitope (mouse monoclonal HA.11 antibody 16B12, Covance Research Products), 6XHis epitope (mouse monoclonal anti-6XHis P5A11 antibody for WB, Biolegend; mouse monoclonal anti-His AD1.10 antibody for IP, Santa Cruz Biotechnology), SUMO-1 (rabbit monoclonal anti-SUMO-1, Millipore), SUMO-2/3 (rabbit polyclonal anti-SUMO-2/3, Millipore), Ubc9 (rabbit monoclonal antibody anti-Ubc9, Cell Signaling), acetyllysine (rabbit polyclonal anti-acetyllysine, ABCAM), GAPDH (rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase antibody, Santa Cruz Biotechnologies) were used for immunoprecipitation and/or immunoblotting.

Techniques: Mutagenesis, Construct, Transfection, Plasmid Preparation, Lysis, Immunoprecipitation, Expressing