Journal: PLoS ONE

Article Title: GCN2 Has Inhibitory Effect on Human Immunodeficiency Virus-1 Protein Synthesis and Is Cleaved upon Viral Infection

doi: 10.1371/journal.pone.0047272

Figure Lengend Snippet: (A) BHK-21 cells were transfected by electroporation with in vitro transcribed capped Sindbis virus RNAs from plasmids pT7SV-HIV-1PR (C+HIV-1 Pro ), pT7SV-2Apro (C+2A Pro ), pT7SVwt (C) for the expression of HIV-1 and PV-2A proteases. After 4 h cells were metabolically labeled with [ 35 S]-Met-Cys. Equivalent amounts of total protein were subjected to 12% SDS-PAGE, transferred to PDVF membranes and subjected to autoradiography (lower panel) to visualize protease expression. The membranes were then probed with specific antisera for detection of endogenous GCN2 and eIF4GI as indicated. (B) COS-7 cells were subjected to coupled infection/DNA transfection with recombinant vaccinia virus (vvT7), pTM1 (−) or pTM1-derived plasmid encoding HIV-1 Pro and pcDNA3.1/Myc-His plasmid, empty (−) or encoding human or mouse GCN2. At 18 h post-transfection cells were lysed and equivalent amounts of total protein were analyzed by western blot using a specific antiserum for detection of GCN2. (C) Affinity purified mouse or human GCN2 was incubated for 3 h at 30°C in the absence or the presence of HIV-1 or HIV-2 proteases. Incubation was stopped by addition of SDS-PAGE sample buffer and proteins were analyzed by western blot using a specific antiserum for detection of GCN2. (D) HeLa cell-free extracts were incubated with recombinant HIV-1 Pro in the absence or the presence of 2.5 µM SQ for the indicated times. Proteins were analyzed by western blot using specific antisera for detection of GCN2, eIF4GI, PABP or actin as indicated. (E) Purified mouse and human GCN2 GST-fusion proteins were incubated in the presence of recombinant HIV-1 Pro for 3 h at 30°C. Proteins were resolved in SDS-PAGE and stained with Coomassie blue. Shown in the lower box is the GCN2 sequence obtained by Edman degradation of the indicated C-terminal fragments. Results are representative of at least three independent experiments.

Article Snippet: For purification of GST fusion proteins, Escherichia coli BL21 cells were transformed with plasmids pGST-mGCN2 (419–1024) and pGST-hGCN2 (525–1024), lysed by sonication, and the fusion proteins were then purified as previously described using glutathione-sepharose (GE Healthcare).

Techniques: Transfection, Electroporation, In Vitro, Expressing, Metabolic Labelling, Labeling, SDS Page, Autoradiography, Infection, Recombinant, Derivative Assay, Plasmid Preparation, Western Blot, Affinity Purification, Incubation, Purification, Staining, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Slc35c2 is primarily localized to Golgi in rat liver. A, a new polyclonal Ab against mouse and human Slc35c2 was tested on lysates of CHO cells expressing empty vector, mouse Slc35c1-Myc, mouse Slc35c2 with low transfection efficiency (C2(low)), or mouse Slc35c2-Myc and analyzed by Western blotting using anti-Slc35c2 C-terminal peptide antibody or anti-Myc. B, cell lysates from CHO cells transiently transfected with mouse Slc35c2 were treated with Endo H or PNGase F and analyzed by Western blotting using anti-Slc35c2 pAb. The membrane was stripped and re-probed with anti-Pofut1 C-terminal peptide pAb. Western blotting was performed using the Odyssey Infrared Imaging System. C, rat liver ER and Golgi fractions were assayed by immunoblotting using anti-Slc35c2 peptide or anti-PDI (ER marker) or anti-GM130 (Golgi marker) or anti-Pofut1 peptide antibodies. Western blotting was performed using the Odyssey Infrared Imaging System.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Membrane, Imaging, Marker

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Slc35c2 inhibits some fucosylation. A, LEC11B cells stably expressing CHO Slc35c2 (C2; black line) or empty vector (Vec; gray line) were assayed for binding of mouse anti-SSEA-1 antibody by flow cytometry. The shaded profile is the secondary antibody alone. B and C, CHO cells transiently transfected with cDNAs encoding CHO Fut6B (B) or Fut9 (C) together with CHO Slc35c2 cDNA (C2; black line) or empty vector (Vec; gray line) were assayed for binding of mouse anti-SSEA-1 antibody by flow cytometry. The shaded profile is the secondary antibody alone. D, binding of FITC-conjugated PSA as in A. The shaded profile is unstained LEC11B cells.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Binding Assay, Flow Cytometry, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Slc35c2 enhances O-fucosylation of a Notch1 fragment. A, Notch1 EGF11–15 was transfected into LEC11B CHO cells stably expressing CHO Slc35c2 or empty vector. Cells were metabolically labeled with l-[3H]fucose, and Notch1 EGF11–15 harvested by Ni2+ beads was analyzed by Western blot with anti-Myc mAb and autoradiography to detect [3H]fucose. B, blots were quantitated by NIH Image J and the relative ratios of [3H]fucose incorporated versus Myc signal are plotted. Error bar, S.E. (n = 3 independent experiments). Slc35c1−/− MEFs were transiently transfected with mouse Slc35c2 cDNA (C2; black line) or mouse Slc35c1 cDNA (C1; dashed line) or both Slc35c1 and Slc35c2 cDNA (C1+C2; dotted line) and analyzed for binding of biotinylated-AAL (C) or FITC-L-PHA (D). The shaded profile is untransfected Slc35c1−/− MEFs for biotinylated-AAL or unstained Slc35c1−/− MEFs for FITC-conjugated L-PHA, respectively.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Transfection, Stable Transfection, Expressing, Plasmid Preparation, Metabolic Labelling, Labeling, Western Blot, Autoradiography, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Knockdown of Slc35c2 reduces Notch1 EGF11–15 fucosylation. A, RNA from CHO cells stably expressing shRNA targeting the CHO Slc35c2 3′ UTR was analyzed by semi-quantitative RT-PCR for Slc35c2 and GAPDH transcripts. B, flow cytometric analysis demonstrated equivalent binding of FITC-PSA to Slc35c2 knockdown cells (KD1, black line; KD2, dashed line) and vector control (Control; gray line). The shaded profile is unstained CHO cells. C, mouse Notch1 EGF11–15 expressed in Slc35c2-KD1 or vector control cells was labeled with l-[3H]fucose, collected on Ni2+ beads, and analyzed by Western blot using anti-Myc mAb and fluorography. D, NIH Image J was used to quantitate blots and the relative ratios of [3H]fucose per Notch1 fragment normalized to vector control were plotted. Error bar, S.E. (n = 3 independent experiments); **, p < 0.01, based on the two-tailed Student's t test.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Knockdown, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Control, Labeling, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Knockdown of Slc35c2 reduces ligand-induced Notch signaling. A, Notch signaling in Slc35c2 knockdown lines (KD1 and KD2) and vector control (Control) using the TP1-luciferase and Renilla luciferase reporter assay. Activation of Notch signaling by Delta1/L or Jagged1/L was determined by the ratio of firefly:Renilla luciferase activities. The values are the average of duplicates from one representative experiment. B, relative activation of Notch signaling in Slc35c2-KD1 and -KD2 normalized to vector control. Error bars, S.E. (n = 4 independent experiments performed in duplicate); **, p < 0.01; ***, p < 0.001, based on the two-tailed Student's t test. C, mouse Notch1 with a C-terminal Myc tag (N1-Myc) was transiently transfected into Slc35c2 knockdown lines (KD1 and KD2) and vector control cells (Control). Delta1-induced Notch1 activation was analyzed by Western blot using N1Val1744 antibody and anti-Myc mAb. Activated Notch1 intracellular domains (N1 ICD) from both endogenous and exogenous N1 were detected. Results of a representative experiment are shown. D, the experiment in C was performed in the presence of 2 μm GSI L-685,458 or DMSO in co-culture medium with Delta1/L cells or control ligand cells (L). Both endogenous and introduced N1 ICDs were blocked by the GSI. E, relative expression of activated Notch1. The quantitation of signals from gels similar to C was performed using NIH Image J. The ratio of activated endogenous Notch1 to loading control (either β-actin or nonspecific band) was normalized to control; the ratio of activated exogenous Notch1 to transfected Notch1 was normalized to control. For endogenous N1 ICD, error bar represent S.E. (n = 4 independent experiments), ***, p < 0.001; for exogenous N1 ICD. Error bars represent S.E. (KD1, n = 5 independent experiments; KD2, n = 4 independent experiments); *, p < 0.05; **, p < 0.01, based on the two-tailed Student's t test.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Knockdown, Plasmid Preparation, Control, Luciferase, Reporter Assay, Activation Assay, Two Tailed Test, Transfection, Western Blot, Co-Culture Assay, Expressing, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Slc35c2 subcellular localization in IEC and HeLa cells. Expression of Slc35c2 in fixed rat IEC (A–F) or HeLa cells (G–N). Transfected Slc35c2 was detected in B, H, J, and L with anti-Slc35c2 pAb; Slc35c2-Myc was detected in D, F, and N with anti-Myc mAb. Other antibodies included anti-PDI (ER marker), anti-ERGIC-53 (ERGIC marker), anti-GM130 (cis-Golgi marker), anti-ManII (medial-Golgi marker), and anti-β4GalT1 (trans-Golgi marker). Images were taken using a Leica SP2 AOBS confocal microscope. Scale bars, 10 μm.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Expressing, Transfection, Marker, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells *

doi: 10.1074/jbc.M110.126003

Figure Lengend Snippet: Quantitation of mouse Slc35c2 subcellular localization after transient transfection IEC or HeLa cells were transiently transfected with mouse Slc35c2 cDNA with or without a C-terminal Myc tag. 100 transfected cells were counted to determine subcellular localization in the secretory pathway.

Article Snippet: An Slc35c2 C-terminal peptide (GEEEYFVTQGQQ) that is conserved in human, rat, and mouse was used to generate rabbit polyclonal antibodies at Proteintech Group Inc. (Chicago, IL) after keyhole limpet hemocyanin conjugation to the first glycine.

Techniques: Quantitation Assay, Transfection

Journal: The Journal of Cell Biology

Article Title: Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

doi: 10.1083/jcb.201504062

Figure Lengend Snippet: Actinonin impairs the turnover of de novo mitochondrial protein synthesis. (A, left) Standard protocol for metabolic labeling of HEK293 cells after ATP5B ( A-5B ) siRNA knockdown or scrambled control (SC) with a chase. Arrows indicate the position of MT-ATP6 and MT-ATP8. (right) SDS-PAGE immunoblot of whole cell lysates with representative antibodies. (B) Modified protocol for extended metabolic labeling of HEK293 cells after ATP5B siRNA knockdown for a 3-h pulse and chase in the indicated conditions (Act, actinonin; E, ethanol). (C) Immunoblot of whole cell lysates of ATP5B siRNA knockdown treated with actinonin or ethanol for 6 h. (D) HEK293 cells metabolically labeled for 45 min with [ 35 S]methionine/cysteine with and without actinonin and then precipitated with CTAB. Each sample was split equally in two with one half treated with RNase A before addition of CTAB. A representative image of three independent experiments is shown.

Article Snippet: Rabbit anti-Mrpl13 was a gift from N. Göran Larsson ( ); from the Proteintech Group, all antibodies were raised in rabbits: anti-AFG3L2 (14631-1-AP), anti-Atp5B (17247-1-AP), anti-ClpP (15698-1AP), anti-Mrpl11 (15543-1-AP), anti-Mrpl23 (11706-1-AP), anti-Mrpl24 (16224-1-AP), anti-Mrpl44 (16394-1-AP), anti-Mrps18b (16139-1-AP), anti-Mrps27 (17280-1-AP), and anti-Yme1l (11510-1-AP); from Sigma-Aldrich, rabbit anti-LonP1 (HPA002192), Mrpl14 (SAB4502786), and anti-HA (H6908); from MitoSciences/Abcam, mouse anti–mt-Co1 (1D6E1A8) and Sdha (C2061); from BD, mouse anti-Opa1 (612606); from Stressgen, rabbit anti-Canx (SPA-860); and from Santa Cruz Biotechnology, Inc., rabbit anti-Tom40 (sc-11414) and goat anti-Hsp60 (sc-1052).

Techniques: Labeling, SDS Page, Western Blot, Modification, Metabolic Labelling

Journal: American Journal of Translational Research

Article Title: CD39 is a promising therapeutic antibody target for the treatment of soft tissue sarcoma

doi:

Figure Lengend Snippet: Overexpression of CD39 in Sarcoma. A. Mass-spectrometric analysis identifies CD39 expression in primary sarcoma samples, but not in non-involved adjacent tissue. B. Representative staining for CD39 on STS. Fibrosarcoma in the peritoneum with IHC score +3 (left panel), fibrosarcoma in the abdominal wall with IHC score +1 (middle panel), CD39-negative normal stroma (right panel). Please note that vasculature is CD39 positive and represents an internal control for all tissue sections. NAT, non-involved adjacent tissue; Scale bar = 50 microns.

Article Snippet: General materials Recombinant His-tagged human CD39 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Over Expression, Expressing, Staining

Journal: American Journal of Translational Research

Article Title: CD39 is a promising therapeutic antibody target for the treatment of soft tissue sarcoma

doi:

Figure Lengend Snippet: IHC score of CD39 in various soft tissue sarcomas and normal stroma

Article Snippet: General materials Recombinant His-tagged human CD39 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques:

Journal: American Journal of Translational Research

Article Title: CD39 is a promising therapeutic antibody target for the treatment of soft tissue sarcoma

doi:

Figure Lengend Snippet: Monoclonal Antibody 9-8B Inhibits Enzymatic Activity of CD39. A. Flow-cytometric analysis shows inhibition of ADP-induced platelet aggregation by 9-8B (4 μM) and POM-1 (10 μM). Naïve; untreated platelets, -; rhCD39 (2 μM)+ ATP (1 mM) treated platelets. B. Orthogonal radioactive CD39 assay demonstrates inhibition of ATPase activity on IGN-SRC-004 cells by 9-8B (6 nM) and POM-1 (10 μM). Ectonucleotidase activity is expressed as picomolar of hydrolyzed free 33Pi per minute per 1000 cells. ***P < 0.001.

Article Snippet: General materials Recombinant His-tagged human CD39 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Activity Assay, Inhibition

Journal: American Journal of Translational Research

Article Title: CD39 is a promising therapeutic antibody target for the treatment of soft tissue sarcoma

doi:

Figure Lengend Snippet: Monoclonal Antibody 9-8B Increases Survival of Metastatic Lethal Patient-derived Sarcoma Xenograft Model IGN-SRC-004. Spontanous metastasis of IGN-SRC-004 to (A) lymph nodes after 21 days and (B) lungs after 30 days. (A) Human fibrosarcoma cells are detected in the lymph nodes using AF647-conjugated anti-human CD39 antibody 5-13 (green). Uptake of 2-deoxyglucose indicates metabolically active sites (red). Overlay (yellow) depicts metabolically active human sarcoma cells. White arrows indicate IGN-SRC-004 positive lymph nodes. L; Left, R; Right, Ht; Heart, Lvr; Liver, Kd; Kidney, Spl; Spleen, Int; Intestine. (B) Double-stained immunohistochemistry for human CD39 (brown) and mouse blood vessel marker CD31 (blue) reveals that human sarcoma cells have breached the lung vasculature. Breakdown of the mouse vascular endothelial wall is also observed. Scale bar = 100 microns. (C) Kaplan-Meier analysis of mice implanted with metastatic IGN-SRC-004. Mice with established tumors of 122 mm3 ± 21 mm3 were randomized and treated at 15 mg/kg with either 9-8B or isotype control antibody (n = 38/treatment). Mantel-Cox p < 0.0001 relative to control. Arrow; administration of antibody.

Article Snippet: General materials Recombinant His-tagged human CD39 fusion protein was purchased from Sinobiological (Beijing, China).

Techniques: Derivative Assay, Metabolic Labelling, Staining, Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets

doi: 10.1101/2024.11.17.624030

Figure Lengend Snippet: (A) Representative confocal images of control (Cont) and PLIN2 deficient (PLIN2KD) INS-1 cells metabolically labeled with Bodipy C12 (C12, red) overnight with or without atglistatin (ATGLi) or lalistat 2 (Lali2) followed by staining with Bodipy 493 (green) and DAPI (white). (B) Number and (C) size of Bodipy C12 positive lipid depots. Data from three independent experiments are combined taking the average value of DMSO treated Cont cells as 1. n=18 to 26 cells total in (B), yielding n= 73 to 506 lipid depots in (C). Mean is indicated. Y axis is log 10 for (B) due to wide distribution of data. (D) TG contents corrected for protein contents and expressed taking value for DMSO treated Cont as 1. Mean ± sem, n=4-8. (E, F) Cont and PLIN2KD INS-1 cells metabolically labeled with C12 overnight followed by immunostaining of mitochondria by anti-HSP60 antibody. A part of PLIN2KD cells were incubated with Lali2 overnight. (E) Pearson’s coefficient for C12 and HSP60 was calculated as in methods. n=15. Representative data from four independent experiments. Mean ± sem. (F) Representative confocal image. *; p <0.05 by One-way ANOVA test with posttest by Sidak’s test for B, D, Krusaki-Wallis test for C, and Dunnett’s test for E.

Article Snippet: Mitochondria in INS-1 cells were visualized using mouse anti-HSP60 antibody (66-41-1-lg, Protein Tech, Rosemont, IL) at 1:300.

Techniques: Control, Metabolic Labelling, Labeling, Staining, Immunostaining, Incubation

Journal: bioRxiv

Article Title: Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets

doi: 10.1101/2024.11.17.624030

Figure Lengend Snippet: (A-D) Mitochondria were visualized by anti-HSP60 antibody in control (Cont) and PLIN2 deficient (PLIN2KD) INS-1 cells. A part of PLIN2KD cells were treated with lalistat 2 (Lali2) overnight prior to fixation. (B) Form factor, (C) mitochondrial length, and (D) aspect ratio were obtained as parameters of mitochondrial morphology as in methods. Data combine three independent experiments and expressed by taking the average of Cont as 1 for each experiment. Each dot represents one cell and n= 23 to 29 cells were counted in total. (E-F) Mitochondria (mitochondria-GFP), beta cells (anti-insulin antibody, white) and nuclei (DAPI, blue) were visualized in human islet cells treated with control (Cont) and lenti-shPLIN2 (PLIN2KD) with or without Lali2. (E) Representative images. (F) Form factor and (G) mitochondrial length obtained as in methods in five donor islets. Data were expressed by taking the average value of DMSO treated Cont as 1 for each donor and each dot represents one donor. All scale bars are 10 μm. Data represent mean ± SEM *; p <0.05 by one-way ANOVA. Tukey’s posttest for (B-D). Sidak posttest for (E and F).

Article Snippet: Mitochondria in INS-1 cells were visualized using mouse anti-HSP60 antibody (66-41-1-lg, Protein Tech, Rosemont, IL) at 1:300.

Techniques: Control

Journal: bioRxiv

Article Title: Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets

doi: 10.1101/2024.11.17.624030

Figure Lengend Snippet: (A) Scheme of a lipid droplet (LD)-lysosome split-GFP-based contact site sensor (SPLICS-P2A-LD–LYSO). Livedrop tagged with GFP 1-10 is expressed on the surface of LD and TMEM192 tagged with β 11 is expressed on the surface of lysosome. When they are within 10 nm vicinity, GFP 1-10 and β 11 fuse to form a GFP. (B, C) Number of green dots corrected for cell area was compared between control (Cont) and PLIN2 downregulated (PLIN2KD) INS-1 cells that were transduced by lentivirus expressing SPLICS-P2A-LD–LYSO. n= 8-10 images. (B) Representative image and (C) representative data from four independent experiments. (D, E) RT-qPCR determined the expression of genes indicated in (D) INS-1 cells (n=11 to 12) and (E) human pseudoislets (n=4 to 6 donors) with or without downregulation (KD) of PLIN2. (F-I) Cont and PLIN2KD INS-1 cells were treated with or without 3.5 mM 3MA overnight in the presence of Bodipy C12. Mitochondria were labeled with anti-HSP60 antibody. (F) Pearson’s coefficient for Bodipy C12 and HSP60. Each dot represents one cell; n=11 to 14 cells combined from two independent experiments. (G) Form factor, (H) mitochondria length, and (I) aspect ratio in Cont and PLIN2KD INS-1 cells expressed taking the average of Cont as 1. Each dot represents one cell and n=13 to 16 cells from two independent experiments are combined. (J, K) Western blot comparing LC3I and LC3II abundance in Cont and PLIN2KD INS-1 cells treated with or without chloroquine (CQ). (J) Representative blot and (K) the ratio of LC3II/I from densitometry obtained from three independent experiments and is expressed taking the average of Cont without CQ as 1. n=8. Data are mean ± SEM. *; p <0.05 by student’s t-test for (C-E, K), and One-way ANOVA for the rest.

Article Snippet: Mitochondria in INS-1 cells were visualized using mouse anti-HSP60 antibody (66-41-1-lg, Protein Tech, Rosemont, IL) at 1:300.

Techniques: Control, Expressing, Quantitative RT-PCR, Labeling, Western Blot

Journal: bioRxiv

Article Title: Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets

doi: 10.1101/2024.11.17.624030

Figure Lengend Snippet: (A) Two potential pathways by which FA are transferred from lysosomes to mitochondria. On the left, FA are secreted to the medium by the lysosome and taken up by the cells prior to entering mitochondria as shown in serum-starved hepatocytes. This step was shown to be inhibited by either binding released FA to fatty acid-free BSA (FFBSA) or by blocking FA reentry using FTAP2 inhibitor CB16.2. On the right, FA are directly transferred from lysosome to mitochondria through contacts. (B-D) Control (Cont) and PLIN2 downregulated (PLIN2KD) INS-1 cells were labeled with Bodipy C12 (C12) overnight and chased for 4 h to measure the secretion of C12 as described in methods. Cont and PLIN2KD INS-1 cells were fixed and immunostained for mitochondria by anti-HSP antibody. (B) C12 in the medium at the end of 4 h was corrected for protein contents in cell lysate and expressed taking the average of Cont as 1. n=6 combined from three independent experiments each in duplicates. (C) Pearson’s coefficient for C12 and HSP60 and (D) mitochondria length in Cont and PLIN2KD cells treated with or without FFBSA or CB16.2. n=5 to 8 images for (C) and 6 to 8 images for (D), representative data of two independent experiments. (E) Representative images and (F) contact number between lysosome marked by CellLight Lysosomes-GFP (green) and mitochondria marked by MitoTracker deep red in Cont and PLIN2KD INS-1 cells. n= 12 to 15 images. Data is representative of three independent experiments. (G) Cont and PLIN2 KD INS-1 cells were cultured with or without lalistat 2 (Lali2) for 16 h and insulin secretion was measured by static incubation. Data were corrected for protein contents and expressed taking the average of Cont at 2.5 mM glucose as 1. n=12 combining four independent experiments. (H) Diagram showing FA trafficking between LD, lysosome and mitochondria in PLIN2 KD beta cells with or without Lali2. Data represent mean ± SEM. *; p <0.05 by one-way ANOVA for (C, D, G) or Student’s t-test for (F). ns: not significant.

Article Snippet: Mitochondria in INS-1 cells were visualized using mouse anti-HSP60 antibody (66-41-1-lg, Protein Tech, Rosemont, IL) at 1:300.

Techniques: Binding Assay, Blocking Assay, Control, Labeling, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Lipid droplet protein Perilipin 2 is critical for the regulation of insulin secretion through beta cell lipophagy and glucagon expression in pancreatic islets

doi: 10.1101/2024.11.17.624030

Figure Lengend Snippet: (A) INS-1 cells were cultured in RPMI medium containing 11.1 mM glucose (Cont) or 20 mM glucose + 0.16 mM oleic acid (OA) + 0.32 mM palmitic acid (PA)(GLT) in the presence or absence of 20 μM lalistat 2 (Lali2) for 48 h. Triglycerides (TG) contents at the end of incubation was measured and corrected for protein contents. n=6, combining duplicates from three independent experiments. (B) Representative images of Bodipy C12 (C12) and HSP60, (C) Pearson’s coefficient for C12 and HSP60, (D) representative image of mitochondria, (E) form factor, and (F) mitochondria length of Cont and GLT INS-1 cells treated for overnight with or without Lali2 in the presence of C12. After fixation, mitochondria were visualized by anti-HSP antibody. n= 28 to 32 images from three independent experiments. (G) Representative image, (H) form factor and (I) mitochondria length of human beta cells cultured in CMRL1066 containing 5.5 mM glucose (Cont) or CMRL1066 at 20 mM glucose + 0.16 mM OA+ 0.32 mM PA (GLT) with or without 10 μM Lali2 overnight. Mitochondria were visualized by CellLight Mitochondria-GFP. Beta cell border identified by anti-insulin antibody is indicated by dotted line. Each dot in figures represents the average of values from each donor, n= 4 donors. (J-M) Human islets were cultured overnight in CMRL1066 medium containing 5.5 mM glucose (Cont) or 12.5 mM glucose + 0.8 mM OA+ 0.16 mM PA (GLT) with or without 10 μM Lali2 overnight. Insulin secretion at 2.8 mM glucose (basal) and 16.8 mM glucose was measured as in methods. (J) Total insulin contents per IEQ, (K) basal insulin secretion, (L) the increment of insulin secretion at 16.8 mM glucose, and (M) stimulation index were expressed taking Cont without Lali2 for each donor as 1. n=6 donors. Data represent mean ± SEM *; p <0.05 by one-way ANOVA (A-L) or Student’s t-test (M). n.s: not significant

Article Snippet: Mitochondria in INS-1 cells were visualized using mouse anti-HSP60 antibody (66-41-1-lg, Protein Tech, Rosemont, IL) at 1:300.

Techniques: Cell Culture, Incubation

Journal: The Journal of Cell Biology

Article Title: Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

doi: 10.1083/jcb.201504062

Figure Lengend Snippet: Loss of AFG3L2 induces aberrant accumulation of MT-ATP6. (A) Immunoblot of HEK293 whole cell lysates after AFG3L2 knockdown with two independent siRNAs compared with scrambled control (sc). The asterisk indicates unspecific signal detected with the AFG3L2 antibody. (B) Immunoblotting of sucrose density gradient fractions of AFG3L2 siRNA. The asterisks indicate unspecific signal in fractions 1 and 2 detected only with MRPS18b antibody with TCA precipitated samples. (C, left) A 30-min 35 S metabolic labeling of cells in A. The arrow indicates MT-ATP6. (right) quantification of the signal intensity of MT-ATP6 relative to MT-CO1 in the same sample for two independent biological experiments of #2 AFG3L2 siRNA and scrambled control (sc). (D) HEK293 cells treated with the indicated siRNA metabolically labeled for 45 min with [ 35 S] methionine/cysteine in the presence of anisomysin and then precipitated with CTAB. Each sample was split equally in two with one half treated with RNase A before addition of CTAB. A representative image of independent biological experiments is shown. a.u., arbitrary units.

Article Snippet: Rabbit anti-Mrpl13 was a gift from N. Göran Larsson ( ); from the Proteintech Group, all antibodies were raised in rabbits: anti-AFG3L2 (14631-1-AP), anti-Atp5B (17247-1-AP), anti-ClpP (15698-1AP), anti-Mrpl11 (15543-1-AP), anti-Mrpl23 (11706-1-AP), anti-Mrpl24 (16224-1-AP), anti-Mrpl44 (16394-1-AP), anti-Mrps18b (16139-1-AP), anti-Mrps27 (17280-1-AP), and anti-Yme1l (11510-1-AP); from Sigma-Aldrich, rabbit anti-LonP1 (HPA002192), Mrpl14 (SAB4502786), and anti-HA (H6908); from MitoSciences/Abcam, mouse anti–mt-Co1 (1D6E1A8) and Sdha (C2061); from BD, mouse anti-Opa1 (612606); from Stressgen, rabbit anti-Canx (SPA-860); and from Santa Cruz Biotechnology, Inc., rabbit anti-Tom40 (sc-11414) and goat anti-Hsp60 (sc-1052).

Techniques: Western Blot, Labeling, Metabolic Labelling

Journal: The Journal of Cell Biology

Article Title: Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

doi: 10.1083/jcb.201504062

Figure Lengend Snippet: Overexpression of AFG3L2 suppresses actinonin effects on translation and OPA1 processing. (A) Growth curve of U2OS cells in actinonin after treatment with the indicated siRNA for 5 d. The data represent mean ± SD; n = 3. (B) A representative, extended 3-h 35 S metabolic labeling in the presence or absence of actinonin after a 5-d siRNA knockdown of AFG3L2 in HEK293 cells. The asterisks represent aberrant translation products. (C) Quantification of the signal intensity of MT-ATP6 relative to MT-CO1 in B from independent biological experiments. Data represent mean ± SD; n = 4. (D, left) Quantification of the signal intensity of MT-ATP6 relative to MT-CO1 from 35 S metabolic labeling from four independent biological experiments with AFG3L2 transient overexpression in HEK293 cells treated with different doses of actinonin for 3 h. Data represent mean ± SD; n = 4. (right) An immunoblot of cells. The asterisk is a nonspecific band detected with the AFG3L2 antibody. (E) An immunoblot of HEK293 cells treated with the indicated siRNA and actinonin doses. (F, left) A representative immunoblot of HEK293 cells treated with the indicated siRNA for 5 d with and without chloramphenicol (CA) or cycloheximide (CHX) for the indicated times. (right) Three panels of data generated from the same samples, only separated on different SDS-PAGE and transferred to independent membranes. a.u., arbitrary units.

Article Snippet: Rabbit anti-Mrpl13 was a gift from N. Göran Larsson ( ); from the Proteintech Group, all antibodies were raised in rabbits: anti-AFG3L2 (14631-1-AP), anti-Atp5B (17247-1-AP), anti-ClpP (15698-1AP), anti-Mrpl11 (15543-1-AP), anti-Mrpl23 (11706-1-AP), anti-Mrpl24 (16224-1-AP), anti-Mrpl44 (16394-1-AP), anti-Mrps18b (16139-1-AP), anti-Mrps27 (17280-1-AP), and anti-Yme1l (11510-1-AP); from Sigma-Aldrich, rabbit anti-LonP1 (HPA002192), Mrpl14 (SAB4502786), and anti-HA (H6908); from MitoSciences/Abcam, mouse anti–mt-Co1 (1D6E1A8) and Sdha (C2061); from BD, mouse anti-Opa1 (612606); from Stressgen, rabbit anti-Canx (SPA-860); and from Santa Cruz Biotechnology, Inc., rabbit anti-Tom40 (sc-11414) and goat anti-Hsp60 (sc-1052).

Techniques: Over Expression, Labeling, Western Blot, Generated, SDS Page

Journal: Nature Communications

Article Title: Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

doi: 10.1038/s41467-022-31876-2

Figure Lengend Snippet: a 5-EC RNABPP workflow. Cells are metabolically labeled with 5-EC and crosslinked RNA-protein complexes are isolated by oligo(dT)-based enrichment. Enriched RNA-protein complexes are digested with RNase and then analyzed by mass spectrometry-based proteomics. b Volcano plot showing the enrichment of 5-EC reactive proteins. Experiment was performed independently in triplicate and protein abundance was quantified by spectral counting. Multiple student’s unpaired two-sided t-tests were used to assess statistical significance. c Western blot validation of ALKBH1, TET2, and MMP8. 293 Flp-In cells overexpressing the respective FLAG-tagged protein were treated with 1 mM 5-EC or DMSO and subjected to the RNABPP workflow before western blot analysis. Experiments were performed in triplicate with similar results. Source data are provided as a Source Data file.

Article Snippet: KO was confirmed by genomic PCR and western blot with mouse β-actin antibody (Cell Signaling, 1:1000, as a loading control) and either rabbit anti-ALKBH1 (Abcam #128895, 1:2000), anti-TET2 (Novus #NBP2-32104, 0.2 µg/mL) or anti-NSUN2 (Proteintech, #20853-1-AP 1:2000) antibodies.

Techniques: Metabolic Labelling, Labeling, Isolation, Mass Spectrometry, Western Blot

Journal: Nature Communications

Article Title: Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

doi: 10.1038/s41467-022-31876-2

Figure Lengend Snippet: a Strategy for mRNA isolation and LC-QQQ-MS quantification of modified nucleosides in HEK 293T wild-type (WT) and KO cells. Total RNA is isolated and subjected to two rounds of poly(A) enrichment, followed by rRNA and small RNA depletion. The mRNA is then digested to nucleosides and analyzed by LC-QQQ-MS. b Quantification of hm 5 C in total RNA from WT and ALKBH KO cell lines. p-values: WT vs. KO #1, p = 0.0000106; WT vs. KO #2, p = 0.0000103. c Quantification of f 5 C in total and mRNA from WT and ALKBH1 KO cell lines. p -values: WT vs. KO #1 in total RNA, 0.000004; in small RNA, 0.000001; WT vs. KO #2 in total RNA, 0.000007; in small RNA, 0.000036. d Quantification of f 5 C in small RNA from WT and ALKBH1 KO cells. p -values: WT vs KO in total RNA, p = 0.000109; in small RNA, p = 0.00000013. e Quantification of f 5 C in total and mRNA from WT and TET2 KO cell lines. p -values: WT vs. KO #1 in total RNA, 0.003594; in small RNA, 0.001040; WT vs. KO #2 in total RNA, 0.002192; in small RNA, 0.049386. f Quantification of f 5 C in total, mRNA, and small RNA from WT and NSUN2 KO cells. p -values: WT vs KO in total RNA, 0.285820; in mRNA, 0.00088; in small RNA; 0.000017. g Quantification of f 5 C in total, mRNA, and small RNA from WT and NSUN3 KO cells. p-values: WT vs KO in total RNA, 0.000007; in mRNA, 0.001198; in small RNA; 0.291189. Three independent biological replicates were analyzed, except in f and g , where n = 6 in total RNA measurements. Data represent mean values ± s.d. An unpaired t test (two-tailed) was used to measure the statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: KO was confirmed by genomic PCR and western blot with mouse β-actin antibody (Cell Signaling, 1:1000, as a loading control) and either rabbit anti-ALKBH1 (Abcam #128895, 1:2000), anti-TET2 (Novus #NBP2-32104, 0.2 µg/mL) or anti-NSUN2 (Proteintech, #20853-1-AP 1:2000) antibodies.

Techniques: Isolation, Modification, Two Tailed Test

Journal: PloS one

Article Title: Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

doi: 10.1371/journal.pone.0011327

Figure Lengend Snippet: Figure 2. rIDE enhances VZV infectivity at an early stage of infection and increases the stability of virus. (A) Cell-free VZV (ROka-LacZ) virus was pre-treated with buffer, bovine serum albumin (BSA), rIDE (15 mg/ml), or denatured rIDE (IDE treated with acid and then base to a final neutral pH) at 37uC for 15 min before infecting melanoma cells. Infected cells were stained with X-gal at 3 days after infection to identify the number of VZV-positive foci. The experiment was repeated 10 times and a representative experiment is shown. For the 5th bar, cells were infected with the same amount of virus as in bars 1-4, and rIDE was then added to the cells 4 hours post-infection. For the last two bars, the virus was incubated with rIDE or buffer at 37uC for 15 min and kept at 25uC for 18 hrs before infecting MeWo cells. (B) Cell-free ROka-LacZ was pre-incubated with increasing concentrations of rIDE (shown in mg/ml) at 37uC for 15 min and infected cells were stained with X-gal. The experiment was performed twice and one result is shown. The control proteins, P7.5 and BZLF2, were used at a concentration of 20 mg/ml. (C) Cell-free VZV (ROka-LacZ) was pre-incubated with rIDE, buffer, or filtrate passing through the rIDE purification column under the indicated conditions and infected cells were stained with X-gal at 4 days post-infection. The filtrate (control) consists of HA peptide which was used to elute rIDE from the purification column. (D) Cell-free ROka-LacZ virus was incubated with buffer, bovine serum albumen (BSA), filtrate that passed through without binding to a rIDE purification column, or rIDE for 18 hrs at 25uC before infecting cells and staining with X-gal. The experiment was performed 3 times, and a representative result is shown. doi:10.1371/journal.pone.0011327.g002

Article Snippet: Cells and viruses Human melanoma cells (MeWo [61], from Charles Grose, University of Iowa) and fibroblast cells (MRC-5, American Type Culture Collection, Manassas, VA) were grown in Minimum Essential Medium with 10% fetal bovine serum (FBS).

Techniques: Infection, Virus, Staining, Incubation, Control, Concentration Assay, Purification, Binding Assay

Journal: PloS one

Article Title: Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

doi: 10.1371/journal.pone.0011327

Figure Lengend Snippet: Figure 4. rIDE promotes VZV internalization during entry and cell-to-cell spread of virus. (A) Cell free ROka-lacZ was incubated with P7.5 control protein or rIDE for 30 min at 37uC, the mixture was incubated with cells for 60 min on ice to allow binding followed by 90 min at 37uC for entry, and DNA was then extracted from the cells and the copy number of intracellular VZV genomes was determined by real-time PCR. The ratio of VZV ORF 62 DNA to cellular RNaseP DNA is shown on the y axis. The experiment was performed three times and a representative result is shown. (B) Melanoma cells were infected with ROka-lacZ, 2.5 hrs later the inoculum was removed by washing with PBS, rIDE was added to the cells, and 3 days later the cells were stained with X-gal. Blue VZV-infected foci were photographed at a magnification 40X and plaque sizes were measured with ImageJ software as previously described [3]. N is the number of plaques measured. (C) Cell-free VZV from a commercial lot of zoster vaccine was incubated with rIDE for 30 min, melanoma cells were infected with the mixture for 4 days, and the cells were fixed and stained with anti-gE monoclonal antibody and infected foci were visualized by fluorescence microscopy. The graph represents pooled data from three independent experiments. Vertical lines indicate standard deviations. doi:10.1371/journal.pone.0011327.g004

Article Snippet: Cells and viruses Human melanoma cells (MeWo [61], from Charles Grose, University of Iowa) and fibroblast cells (MRC-5, American Type Culture Collection, Manassas, VA) were grown in Minimum Essential Medium with 10% fetal bovine serum (FBS).

Techniques: Virus, Incubation, Control, Binding Assay, Real-time Polymerase Chain Reaction, Infection, Staining, Software, Fluorescence, Microscopy

Journal: PloS one

Article Title: Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

doi: 10.1371/journal.pone.0011327

Figure Lengend Snippet: Figure 7. VZV mutant lacking the IDE binding domain of gE shows reduced fusogenicity by membrane fusion assay. (A) Melanoma cells expressing T7 polymerase were infected with VZV ROka, ROka68D32-71, and the cells were incubated with HeLa cells containing the b– galactosidase gene driven by the T7 promoter or a control GFP plasmid for 20 hr. The cells were lysed, incubated with chlorophenol red-b-D- galactopyranoside, and b–galactosidase activity was measured using a spectrophotometer at OD570 nm. The experiment was also performed at 16 hr and 24 hr and similar results were obtained. Vertical lines indicate standard deviations. (B) Human melanoma cells expressing T7 polymerase were infected with the same MOI of ROka-GFP or ROka68D32-71-GFP, and cells were co-incubated with HeLa cells containing the b–galactosidase gene driven by the T7 promoter for 16 hrs. The cells were lysed, incubated with chlorophenol red-b-D-galactopyranoside, and b–galactosidase activity was measured using a spectrophotometer at OD570 nm. The experiment was performed twice with similar results. (P,0.001 for ROka-GFP vs. ROka68D32-71-GFP, Student t test). Vertical lines indicate standard deviations. doi:10.1371/journal.pone.0011327.g007

Article Snippet: Cells and viruses Human melanoma cells (MeWo [61], from Charles Grose, University of Iowa) and fibroblast cells (MRC-5, American Type Culture Collection, Manassas, VA) were grown in Minimum Essential Medium with 10% fetal bovine serum (FBS).

Techniques: Mutagenesis, Binding Assay, Membrane, Single Vesicle Fusion Assay, Expressing, Infection, Incubation, Control, Plasmid Preparation, Activity Assay, Spectrophotometry

Journal: PloS one

Article Title: Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

doi: 10.1371/journal.pone.0011327

Figure Lengend Snippet: Figure 10. Deletion of IDE binding domain on gE (amino acids 32–71) does not affect myristylation of gE. ROka or ROka68D32-71 infected cells were radiolabeled with 3H-myristic acid and immunopre- cipitated with anti-gE antibody. doi:10.1371/journal.pone.0011327.g010 Figure 11. Treatment of VZV with rIDE does not increase virus binding. POka (parental Oka VZV) or mock infected MRC-5 cells were metabolically labeled with 3H-thymidine at 0.25 mCi/ml for 36 hr beginning at 8 hr post-infection, and cell-free virus was prepared by sonication as described above. The virus was then pre-incubated with rIDE or control proteins at 37uC for 30 min before addition to human melanoma cells on ice for 80 min in the presence of 100 mg/ml of heparin (to block binding through cell surface heparin sulfate). After extensive washing with cold PBS, the cells were lysed and radioactivity bound to cells was measured. The number of radioactive counts per minute for mock-infected MRC-5 cells was subtracted from the counts per minute for POka-infected cells (P = 0.1 for rIDE vs. BSA or for rIDE vs. BZLF2, Student t test). The data shown are based on two independent experiments. doi:10.1371/journal.pone.0011327.g011

Article Snippet: Cells and viruses Human melanoma cells (MeWo [61], from Charles Grose, University of Iowa) and fibroblast cells (MRC-5, American Type Culture Collection, Manassas, VA) were grown in Minimum Essential Medium with 10% fetal bovine serum (FBS).

Techniques: Binding Assay, Infection, Virus, Metabolic Labelling, Labeling, Sonication, Incubation, Control, Blocking Assay, Radioactivity

Journal:

Article Title: Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

doi: 10.1128/JVI.77.14.7779-7785.2003

Figure Lengend Snippet: Synthesis and processing of mutant and wild-type (WT) glycoproteins. (A) Diagram of the M-PMV TM protein showing the location of the HR1 domain of M-PMV relative to the rest of the glycoprotein. The positions of the primers used to introduce mutations are shown. (B) COS-1 cells were transfected with the pTMT expression vector containing either wild-type or mutant env genes. At 48 h posttransfection, the cells were labeled with [3H]leucine for 30 min and immunoprecipitated with goat anti-M-PMV serum as described in the text. The mutant designations are shown above each lane, and the position of the precursor glycoprotein, Pr86, is indicated to the left. Mock, mock transfected. (C) Processing of the env gene polyprotein precursor protein. Following a 4-h chase in unlabeled medium, Env proteins were immunoprecipitated with goat anti-M-PMV serum and electrophoresed in SDS-PAGE. The positions of the precursor glycoprotein, Pr86, and the cleavage products, gp70 and gp22, are shown. (D) Position of gp22 after longer exposure. (E) Secretion of SU into culture media. Following pulse-chase, the culture media were collected, immunoprecipitated with goat anti-M-PMV serum, and analyzed for the released SU proteins.

Article Snippet: African green monkey kidney (COS-1) cells were obtained from the American Type Culture Collection.

Techniques: Mutagenesis, Introduce, Transfection, Expressing, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Pulse Chase

Journal:

Article Title: Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

doi: 10.1128/JVI.77.14.7779-7785.2003

Figure Lengend Snippet: Expression of envelope glycoprotein mutants in the context of provirus. The mutant env genes in pSARM4 were transfected into COS-1 cells and metabolically labeled as described in Materials and Methods. (A) Cell lysates were immunoprecipitated with goat anti-M-PMV serum and analyzed in SDS-PAGE. The mutant designation is shown above each lane, and the positions of the viral bands are indicated on the left. WT, wild type; Mock, mock transfected. (B and C) Incorporation of mutant glycoprotein into virions. Virus-containing supernatants from metabolically labeled COS-1 cells transfected with either wild-type or mutant pSARM4 constructs were centrifuged through a 25% sucrose cushion. The virus pellets were recovered and immunoprecipitated with goat anti-M-PMV serum (B) or electrophoresed directly (C). The positions of the viral bands are indicated on the left.

Article Snippet: African green monkey kidney (COS-1) cells were obtained from the American Type Culture Collection.

Techniques: Expressing, Mutagenesis, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Virus, Construct

Journal:

Article Title: Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

doi: 10.1128/JVI.77.14.7779-7785.2003

Figure Lengend Snippet: Cell-cell fusion assay of envelope glycoproteins expressed from the pTMT expression vector. COS-1 cells were transfected with the pTMT expression vector containing either the mutant or wild-type (WT) env gene. COS-1 cells expressing glycoproteins were mixed 1:2 with GHOST cells, replated, and analyzed as described in Materials and Methods. (A) Cells were gated on size and then analyzed for expression of GFP by quantitating excitation at 488 nm. (B) The percentage of fused GFP-positive cells was calculated for each mutant and normalized to the wild type as shown. The graph depicts the mean of three independent experiments (± standard deviation). The mutant designation is shown below each histogram.

Article Snippet: African green monkey kidney (COS-1) cells were obtained from the American Type Culture Collection.

Techniques: Cell-Cell Fusion Assay, Expressing, Plasmid Preparation, Transfection, Mutagenesis, Standard Deviation

Journal:

Article Title: Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

doi: 10.1128/JVI.77.14.7779-7785.2003

Figure Lengend Snippet: Single-round infectivity assay of virus. COS-1 cells were cotransfected with pTMT and pMΤΔΕ expression vectors as described in Materials and Methods. Culture medium from cells expressing virus was filtered and normalized for RT activity. The normalized medium was used to infect GHOST cells. (A) Cells were gated on size and granularity and then analyzed for GFP expression. WT, wild type. (B) A total of 3 × 104 cells were analyzed for each construct, and the number of GFP-expressing cells relative to the wild type was plotted. The graph shows the mean from three independent experiments (± standard deviation).

Article Snippet: African green monkey kidney (COS-1) cells were obtained from the American Type Culture Collection.

Techniques: Infection, Virus, Expressing, Activity Assay, Construct, Standard Deviation