Journal: Viruses

Article Title: m 6 A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication

doi: 10.3390/v15061381

Figure Lengend Snippet: GPRC5A interacts with members of the Flotillin family and affects NFκB signalling. ( A ) Proteins with >1.5 enrichment in FLAG immunoprecipitates from GPRC5A-FLAG-overexpressing TREx-BCBL1-Rta cells compared with parental TREx-BCBL1-Rta cells after TMT mass spectrometry analysis (grey box: data points with less than 50 abundance in both latent and lytic cells were excluded). ( B ) STRING interaction map showing proteins with >1.4-fold interaction with GPRC5A in lytic replication compared with latency in TREx-BCBL1-Rta cells after TMT mass spectrometry analysis. ( C ) Immunoprecipitation of GFP or GPRC5A-GFP from transduced latent and lytic TREx-BCBL1-Rta cells showing increased co-immunoprecipitation of FLOT1 in lytic replication. ( D ) Immunofluorescence analysis of FLOT1 localisation in latent and lytic TREx-BCBL1-Rta cells transduced with a GFP control (upper two) or GFP-tagged (lower two) GPRC5A-expressing plasmid. ( E ) Representative Western blots of NFκB, pNFκB, pSTAT, pAKT, pERK and GAPDH protein expression in latent and lytic GPRC5A-depleted cells. ( F ) Densitometry quantification of NFκB and pNFκB immunoblots was performed using ImageJ software, and is shown as a percentage relative to the loading control, GAPDH. ( G ) Luciferase reporter assay from HEK 293Ts co-transfected with GFP or GPRC5A-GFP alongside various described signalling reporters with transcription factor binding sites attached to a luciferase reporter plasmid. Data presented are relative to an internal firefly control. In ( F , G ), data are presented as mean ± SD. * p < 0.01. All repeats are biological (n = 3).

Article Snippet: Antibodies used for immunoblotting: C-Myc (Sigma M4439 1:1000), FLOT1 (CST D2V7J 1:1000, Leiden, Netherlands), FTO (Abcam ab126605 1:5000, Cambridge UK), GAPDH (Proteintech 60004-1-Ig 1:5000, Manchester, UK), GFP (Living Colours 632381 1:1000, Kusatsu, Japan), GPRC5A (Atlas HPA007928 1:500, Broma, Sweden), NFκB (CST D14E12, Leiden, Netherlands), phospho-NFκB (CST 93H1, Leiden, Netherlands), ORF57 (Santa Cruz sc-135747 1:1000, Dallas, TX, USA), ORF65 (CRB crb2005224 1:100, Cambridge, UK), STC1 (Proteintech 20621-1-AP 1:500, Manchester, UK)), WTAP (Abcam ab195380 Rabbit 1:1000, Cambridge, UK). pVSV.G and psPAX2 were gifted by Dr Edwin Chen (University of Westminster, London, UK).

Techniques: Mass Spectrometry, Immunoprecipitation, Immunofluorescence, Transduction, Control, Expressing, Plasmid Preparation, Western Blot, Software, Luciferase, Reporter Assay, Transfection, Binding Assay

Journal: International journal of molecular sciences

Article Title: Long Interspersed Nuclear Element-1 Analytes in Extracellular Vesicles as Tools for Molecular Diagnostics of Non-Small Cell Lung Cancer.

doi: 10.3390/ijms25021169

Figure Lengend Snippet: Figure 1. EV isolation and identification of LINE-1 mRNA and ORF1p EV cargo. (A) LINE-1 element structure, proteins, and RNP. Full length LINE-1 is approximately 6 kb in length and consists of 5′ and 3′ untranslated regions (UTRs) and two proteins, ORF1p (orange) and ORF2p (blue). ORF1 is a nucleic acid binding protein consisting of an alpha helix in between the N- and C- terminal domains (NTC, CTD), with an RNA recognition motif (RRM) near the CTD. Coiled-coil interactions facilitate the formation of higher order multimers and polymers of ORF1p. ORF2p is approximately 150 kDa and contains both endonuclease (EN) and reverse transcriptase (RT) domains. Both ORF1p and ORF2p exhibit strong affinity for the LINE-1 mRNA, and together form ribonucleoproteins (RNPs) containing both the template and enzymatic machinery required for target-site primed reverse transcription. EVs from H520 cells were collected using ultracentrifugation along with unconditioned media (UCM) and EV-free media (EFM). Equal quantities of EV protein, cell lysate protein, and an equivalent volume of UCM and EFM (30 µL) were examined using SDS-PAGE and Western blotting. (B) Nanoparticle tracking analysis was performed to examine the size and abundance of EV isolates. Mean and SE of five measurements shown. (C) The EV markers, ALIX, Flotillin-1, and CD9 were examined in the preparations. The absence of calnexin, an endoplasmic reticulum protein, indicates absence of contaminating proteins. The presence of LINE-1 protein, ORF1p, was examined using an in-house polyclonal antibody. ORF1p forms multimers resistant to denaturation during reducing SDS-PAGE. Unlike cell lysates, an ORF1p dimer is predominant in EVs. EV protein treated with the irreversible reducing agent N-ethylmaleimide (NEM) was included for reference. (D) Ponceau staining was completed to confirm protein loading. (E) H460 cell cultures were treated with a LINE-1 inducer, benzo[a]pyrene (BaP) and the EVs were collected and examined via SDS-PAGE, which resulted in the appearance of the ORF1p dimer. (F) To demonstrate the presence of LINE-1 mRNA within EVs, intact EVs were treated with RNAseA prior to lysis and RNA isolation. Similar treatments were performed with UCM and EFM controls for comparison. β-Actin is shown as a positive control. LINE-1 was normalized to RTC and β-Actin to NTC. Mean, SD shown. N = 2. * FC > 1, above background; p < 0.05. One-tail t-test.

Article Snippet: 2024, 25, 1169 13 of 16 Antibodies to ALIX (CST, 92880), CD9 (CST, 13403), Flotillin-1 (CST, 18634), GAPDH (CST, 2118), and Calnexin (MA5-31501) were obtained commercially via Cell Signaling Technology (Danvers, MA, USA) and ThermoFisher.

Techniques: Isolation, Binding Assay, Reverse Transcription, SDS Page, Western Blot, Staining, Lysis, Comparison, Positive Control