Journal: Cell Host & Microbe

Article Title: Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection

doi: 10.1016/j.chom.2022.10.018

Figure Lengend Snippet:

Article Snippet: Four-fold serial dilutions of mAbs diluted to 2.5 μg/mL in Casein were then added for 2 h. A 1:3000 dilution of HRP-labeled goat anti-rabbit IgG (Jackson Immunoresearch) in Casein was added for 1 h. Up to now, between each step, plates were washed three times with TBS.

Techniques: Virus, Recombinant, Mass Spectrometry, Sequencing, Lysis, Emulsion, Adjuvant, Plasmid Preparation, High Molecular Weight, Marker, Electron Microscopy, Luciferase, Software, Chromatography, Random Hexamer, Luminex, Expressing, Modification, Nano Differential Scanning Fluorimetry, Clinical Proteomics

Journal: Scientific Reports

Article Title: Spermine increases acetylation of tubulins and facilitates autophagic degradation of prion aggregates

doi: 10.1038/s41598-018-28296-y

Figure Lengend Snippet: Prion aggregates traffic via autolysosomes in SMB.s15 cells. All the experiments were performed either with or without 10 μM CQ. ( a ) Immunofluorescence staining using anti PrP antibody BC6 in SMB-PS and SMB.s15 cells (N = 3). ( b ) Representative immunoblot shows PK resistant PrP Sc in SMB.s15 cells (N = 2). ( c ) Representative immunoblot for LC3 and β-tubulin in SMB-PS and SMB.s15 cells, lane 2 & 4 were treated with CQ (N = 3). ( d ) Representative TEM images of SMB-PS and SMB.s15 after CQ treatment, red arrows indicate vesicles with aggregates (N = 2). ( e ) Increased number of vesicles with aggregates in SMB.s15 cells. All SMB.PS cells imaged with TEM showed minimal aggregates (0–5/cell) whereas all SMB.s15 cells images showed increased number of aggregates (5–24/cell). ( f ) WB for PK resistant PrP Sc after CQ treatment in SMB.s15 cells (N = 3). ( g ) Representative image of proteostat dye (aggregates) and LC3 immunofluorescence staining of SMB.s15 cells after CQ treatment compared to untreated control cells (N = 3). ( h ) Significantly increased LC3 stained proteostat positive aggregates in SMB.s15 cells on CQ treatment. ( i ) Representative image of PrP Sc and LC3 immunofluorescence staining of SMB.s15 cells after CQ treatment compared to untreated control cells (N = 5). ( j ) Significantly increased LC3 stained PrP Sc aggregates in SMB.s15 cells on CQ treatment. Scale bar = 10 μm. Respective full length blots are shown in Supplementary Fig. 3. Abbreviations used Chloroquine (CQ), Transmission Electron Microscopy (TEM), proteinase K (PK).

Article Snippet: Following antibodies were used, mouse monoclonal ROS-BC6 (BC6) (anti-prion) 1.3 mg/ml (TSE Resource Centre, Cat no: RC 063 ), rabbit polyclonal anti-LC3 (MBL, PM036) 1:3000 for WB and 1:300 for imaging, anti-acetyl alpha tubulin (6-11B-1) (Santa Cruz biotech, sc-23950) and mouse monoclonal anti- acetylated tubulin (Lys40) (Proteintech, cat no 66200-1-Ig) both used at 1:1000 for WB and 1:300 for imaging, mouse monoclonal anti-DyneinHC (C-5) (Santa Cruz biotech, sc-514579) 1:1000 for WB, rabbit polyclonal anti-Tubb6 (Aviva systems biology, ARP60382_P050) 1:1000 for WB and 1:300 for imaging, rabbit polyclonal anti-HSP47 (Abcam, ab77609), rabbit polyclonal anti-UbAP2L (Thermo scientific, PA5-29520) both 1:300 for imaging, rabbit monoclonal anti-HDAC6 (Abcam ab133493) used at 1:100 for imaging and rabbit polyclonal anti-beta actin (GeneTex, cat no GTX109639), Rabbit Polyclonal ATG5 (Sigma, A0731), used at 1:1000 for WB, secondary antibodies goat anti rabbit 488 (Thermo Fisher Scientific A11034) and goat anti mouse 647 (Thermo Fisher Scientific A21236) were both used at 1:500 for microscopy.

Techniques: Immunofluorescence, Staining, Western Blot, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Spermine increases acetylation of tubulins and facilitates autophagic degradation of prion aggregates

doi: 10.1038/s41598-018-28296-y

Figure Lengend Snippet: Spermine increases the acetylation of proteins involved in retrograde transport. SMB.s15 cells were treated with 5 μM Spermine for 0, 24 and 72 hrs; cytoplasmic extracts were used both for immunoprecipitation (IP) with anti-acetyl lysine antibody and run on WB or run directly on WB. ( a ) Immunoblotting with total alpha tubulin (lane1), two different acetyl-alpha tubulin antibodies (6–11B-1 and Lys40) show increased expression (lane 2 and 3) after 24 hrs spermine treatment, and loading control β-actin (lane 4) both from the cytoplasmic extract as well as from the immunoprecipitation product after IP with acetyl-lysine antibody (lane 5), BAC (lane 5 last row) ( b ) Graph showing relative expression levels of 6-11B-1 and Lys40 compared to β-actin. ( c ) WB shows increased expression of approx. 500 kDa Dynein-HC after 24 and 72 hrs of spermine treatment ( d ) Representative images with acetyl-alpha antibodies (6-11B-1, first lane) (Lys40, second lane) show increased expression on 24 hrs spermine treatment. ( e ) Representative images with increased colocalisation of bright LC3 punctae with acetyl-alpha antibody (6-11B-1) after 24 hrs spermine treatment. Scale bar = 10 μm, N = 3 for LC-MS, N = 2 for WB and immunofluorescence. Respective full length blots are shown in Supplementary Fig. . Abbreviations used: Immunoprecipitation (IP), Western blot (WB), Beads Alone Control (BAC), Liquid chromatography–mass spectrometry (LC-MS).

Article Snippet: Following antibodies were used, mouse monoclonal ROS-BC6 (BC6) (anti-prion) 1.3 mg/ml (TSE Resource Centre, Cat no: RC 063 ), rabbit polyclonal anti-LC3 (MBL, PM036) 1:3000 for WB and 1:300 for imaging, anti-acetyl alpha tubulin (6-11B-1) (Santa Cruz biotech, sc-23950) and mouse monoclonal anti- acetylated tubulin (Lys40) (Proteintech, cat no 66200-1-Ig) both used at 1:1000 for WB and 1:300 for imaging, mouse monoclonal anti-DyneinHC (C-5) (Santa Cruz biotech, sc-514579) 1:1000 for WB, rabbit polyclonal anti-Tubb6 (Aviva systems biology, ARP60382_P050) 1:1000 for WB and 1:300 for imaging, rabbit polyclonal anti-HSP47 (Abcam, ab77609), rabbit polyclonal anti-UbAP2L (Thermo scientific, PA5-29520) both 1:300 for imaging, rabbit monoclonal anti-HDAC6 (Abcam ab133493) used at 1:100 for imaging and rabbit polyclonal anti-beta actin (GeneTex, cat no GTX109639), Rabbit Polyclonal ATG5 (Sigma, A0731), used at 1:1000 for WB, secondary antibodies goat anti rabbit 488 (Thermo Fisher Scientific A11034) and goat anti mouse 647 (Thermo Fisher Scientific A21236) were both used at 1:500 for microscopy.

Techniques: Immunoprecipitation, Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Liquid Chromatography, Mass Spectrometry