Journal: Analytical Chemistry

Article Title: Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Analysis for the Direct Detection of SARS-CoV-2 in Nasopharyngeal Swabs

doi: 10.1021/acs.analchem.1c04328

Figure Lengend Snippet: Purification and concentration of SARS-CoV-2 particles in a nasopharyngeal swab eluate. (A) SDS-PAGE analysis of concentrated SARS-CoV-2 particles. Lane M contained the protein standard. The SARS-CoV-2 suspension (lane 1) was concentrated by each of the three MWCO ultrafiltration cartridges. The concentrated fraction (CF) and the flow-through fraction (FF) were analyzed to check which fraction the virus particles were collected in. Lane 2 contained the CF of 30 kDa MWCO, lane 3 contained the FF of 30 kDa MWCO, lane 4 contained the CF of 100 kDa MWCO, lane 5 contained the FF of 100 kDa MWCO, lane 6 contained the CF of 300 kDa MWCO, and lane 7 contained the FF of 300 kDa MWCO. (B) SDS-PAGE analysis of fractionated proteins in a nasopharyngeal swab eluate. Lane M contained the protein standard. The swab eluate (lane 1) was passed through each of the three molecular weight cutoff (MWCO) ultrafiltration cartridges. The CF and FF were analyzed to check the separated proteins. Lane 2 contained the CF of 30 kDa MWCO, lane 3 contained the FF of 30 kDa MWCO, lane 4 contained the CF of 100 kDa MWCO, lane 5 contained the FF of 100 kDa MWCO, lane 6 contained the CF of 300 kDa MWCO, and lane 7 contained the FF of 300 kDa MWCO. (C) SDS-PAGE analysis of purification steps for SARS-CoV-2 particles from a nasopharyngeal swab eluate. Lane M contained the protein standard. SARS-CoV-2 particles were mixed with a nasopharyngeal swab eluate (lane 1) and fractionated by a 300 kDa MWCO ultrafiltration cartridge. The FF (lane 2) and CF (lane 3) were analyzed to check the separated proteins and virus particles. The CF was further purified by Q Sepharose XL resin to remove contaminating proteins from the nasopharyngeal swab eluate (lane 4). Black arrows and a red arrow indicate contaminating proteins and the SARS-CoV-2 nucleocapsid phosphoprotein, respectively.

Article Snippet: Further purification was performed by mixing the fraction with 50 μL of 2-propanol, then adding 20 μL (suspension volume) of Q Sepharose XL resin (Cytiva, Uppsala, Sweden).

Techniques: Purification, Concentration Assay, SDS Page, Suspension, Virus, Molecular Weight

Journal: Cell reports

Article Title: Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

doi: 10.1016/j.celrep.2023.112807

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ATP agarose , Novus Biologicals , Cat# 510–0002.

Techniques: Virus, Recombinant, Proximity Ligation Assay, Software

Journal: Nature microbiology

Article Title: TRIM23 mediates virus-induced autophagy via activation of TBK1

doi: 10.1038/s41564-017-0017-2

Figure Lengend Snippet: a, Homology models of the TRIM23 ARF domain (residues 402–574) based upon the GTP- and GDP-bound forms of ARF6. The five lysines that comprise the main sites of auto-ubiquitination are shown as blue sticks, with positional differences indicated by arrows (GTP to GDP). The position of K402 is shown only for the GDP-bound form as this residue is absent from the GTP-bound homology model. The active site containing bound nucleotide is indicated on the lower right. b, Quantification of GFP-LC3B puncta in TRIM23 −/− MEF cells transiently transfected with GFP-LC3B together with empty vector, or the indicated FLAG-tagged TRIM23 constructs. 48 h later, cells were stained with anti-FLAG antibody, and GFP-LC3B puncta were quantified in cells positive for anti-FLAG-staining. Results represent the mean LC3B puncta per cell ± SD (n=30). ***p <0.001 (Student’s t -test). c, In vitro GTPase activity of purified FLAG-tagged TRIM23 WT and mutants, or Rab5α (positive control). Purified proteins were incubated with GTP in vitro , followed by measuring GTP hydrolysis using a luciferase-based readout. Results are expressed as mean ± SD (n=3). d, Binding of TRIM23 WT and mutants to GTP-coupled agarose. FLAG-tagged TRIM23 constructs were expressed in transiently transfected HEK293T cells. 48 h later, WCLs were incubated with GTP-agarose and proteins bound to GTP determined by IB with anti-FLAG antibody. e, In vitro GTPase activity of purified FLAG-tagged TRIM23 WT from cells co-expressing HA-tagged Ub WT, or the Ub mutants K27R or K27only. Purified TRIM23 WT was incubated with GTP in vitro, followed by measuring GTP hydrolysis activity as described in (c). Results are expressed as mean ± SD (n=3). *p <0.1 (Student’s t -test). ns, statistically not significant. f, Co-localization of endogenous p62 with FLAG-tagged TRIM23 WT or mutants in transiently transfected HeLa cells, determined by immunostaining with anti-FLAG (TRIM23, green) and anti-p62 (red), followed by confocal microscopy analysis. DAPI, nuclei (blue); Scale bar, 20 µm. Data are representative of one ( a ), or at least two ( b–f ) independent experiments.

Article Snippet: Post-centrifuged lysates were mixed with a ~50% slurry of GTP agarose resin (Novus Biologicals, 505-0001) and incubated for 2 h at 4 °C.

Techniques: Ubiquitin Proteomics, Residue, Transfection, Plasmid Preparation, Construct, Staining, In Vitro, Activity Assay, Purification, Positive Control, Incubation, Luciferase, Binding Assay, Expressing, Immunostaining, Confocal Microscopy