primary antibody (rabbit polyclonal anti-arc)  (Synaptic Systems)

Journal: bioRxiv

Article Title: Retrovirus-Like Gag Protein Arc/Arg3.1 is Involved in Extracellular-Vesicle-Mediated mRNA Transfer between Glioma Cells

doi: 10.1101/2023.04.11.536339

Figure Lengend Snippet: Measurement of Arc/Arg3.1 protein binding affinity to mRNA using biolayer interferometry. (A) The scheme of the measurement procedure, which includes the following steps: 1) incubation of streptavidin (SA) biosensors in PBS to set baseline I; 2) loading of the biosensor with biotinylated mRNA; 3) washing of biotinylated mRNA and the setting baseline II; 4) binding with Arc protein; 5) dissociation of Arc protein. (B) Fitted experimental Arc-mRNA association and dissociation curves shown as means with SD error bars. Calculated values of (C) K on , (D) K off , and (E) apparent K D for Arc protein/ mCherry mRNA and Arc protein/ Arc mRNA shown as means ± SD. * p < 0.05, Mann-Whitney U test.

Article Snippet: The membrane was blocked for 1 h with a blocking buffer (Bio-Rad) under gentle agitation and incubated overnight with antibodies against Arc/Arg3.1 (TA349500, OriGene), CD9 (ab236630, Abcam), CD63 (ab271286, Abcam), CD81 (ab59477, Abcam) or GAPDH (ab8245, Abcam) as a reference.

Techniques: Protein Binding, Incubation, Binding Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Retrovirus-Like Gag Protein Arc/Arg3.1 is Involved in Extracellular-Vesicle-Mediated mRNA Transfer between Glioma Cells

doi: 10.1101/2023.04.11.536339

Figure Lengend Snippet: Characterization of Arc EVs. (A) As determined by NTA analysis, Arc EVs demonstrated two-peak size distribution with average diameter of 130 nm. (B) Besides Arc/Arg3.1 protein, immunoblotting analysis indicated the presence of exosome-specific tetraspanins (CD9, CD63, and CD81) and less amount of GAPDH associated with Arc EVs. (C) qRT-PCR analysis of Arc and GFP mRNA content in EVs obtained from HEK293, WT U87, and Arc-GFP U87 cells. (D) TEM images of EVs, isolated from WT and Arc-overexpressing HEK293, U87, LN229, and U251 cells. The scale bar is 200 nm. Quantitative analysis of TEM images of EVs showed a decrease in the mean diameter of EVs (E) and a significant increase in their production (F) by Arc-overexpressing cells. Data are shown as means ± SD. *** p < 0.001, Mann-Whitney U test.

Article Snippet: The membrane was blocked for 1 h with a blocking buffer (Bio-Rad) under gentle agitation and incubated overnight with antibodies against Arc/Arg3.1 (TA349500, OriGene), CD9 (ab236630, Abcam), CD63 (ab271286, Abcam), CD81 (ab59477, Abcam) or GAPDH (ab8245, Abcam) as a reference.

Techniques: Western Blot, Quantitative RT-PCR, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Retrovirus-Like Gag Protein Arc/Arg3.1 is Involved in Extracellular-Vesicle-Mediated mRNA Transfer between Glioma Cells

doi: 10.1101/2023.04.11.536339

Figure Lengend Snippet: Influence of Arc expression on GFP and RFP mRNA transfer in glioma cell coculture. (A) Immunoblotting evaluation of Arc/Arg3.1 protein expression by Arc KO U87 cells. (B) Evaluation of Arc mRNA expression level in Arc WT, Arc KO, and Arc-GFP U87 cells. (C) A scheme of mRNA transfer in Arc-GFP + Arc KO/RFP U87 and Arc KO/GFP + Arc KO/RFP U87 cell co-cultures. (D) Flow cytometry analysis of GFP- and RFP-expressing U87 cell lines and their co-cultures. (E) It was found that the percentage of GFP and RFP-positive cells in the co-culture of Arc-GFP U87 and Arc KO/RFP U87 cells was 3-fold higher than in Arc KO co-culture. Data are shown as means ± SD. * p < 0.05, Mann-Whitney U test. (F) Images of Arc-GFP + Arc KO/RFP U87 and Arc KO/GFP + Arc KO/RFP U87 cell cocultures after 72-h incubation. White arrows indicate GFP and RFP co-expressing cells. The scale bar is 50 μm.

Article Snippet: The membrane was blocked for 1 h with a blocking buffer (Bio-Rad) under gentle agitation and incubated overnight with antibodies against Arc/Arg3.1 (TA349500, OriGene), CD9 (ab236630, Abcam), CD63 (ab271286, Abcam), CD81 (ab59477, Abcam) or GAPDH (ab8245, Abcam) as a reference.

Techniques: Expressing, Western Blot, Flow Cytometry, Co-Culture Assay, MANN-WHITNEY, Incubation