Journal: The Journal of Neuroscience

Article Title: Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes

doi: 10.1523/JNEUROSCI.2014-22.2023

Figure Lengend Snippet: Drift correction and localization error of QD probes in fixed hippocampal neurons expressing the YFP-tagged GluN1-1a subunit. A , Representative images of hippocampal neurons. Left, Neurons were stained by incubation in 20 n m MitoTracker Deep Red FM marker (catalog #M22426, Thermo Fisher Scientific) for 30 s in Neurobasal medium. Right, Neurons were infected with a lentivirus expressing the synaptic protein tdTomato-Homer1c. B , Schematic diagram depicting the three QD-based probes used in this study. The antiGFP-QD605 probe contains the rabbit anti-GFP IgG antibody combined with a secondary IgG antibody conjugated to QD605. The nanoGFP-QD605 probe contains an anti-GFP nanobody conjugated to QD605, while the nanoGFP-QD525 probe contains the anti-GFP nanobody conjugated to QD525. C , Negatively stained samples of both nanoGFP-QD probes were imaged at a magnification of 60,000× and a pixel size of 1.939 Å/px by TEM. Measured average diameters of both nanoGFP-QD probes ± SEM were 16.2 ± 0.4 nm (nanoGFP-QD525) and 20.4 ± 0.6 nm (nanoGFP-QD605; n ≥ 30). D , Example of drift estimated from multiple QD trajectories of the YFP-GluN1-1a subunit. E , The corresponding xy drift path. F , Example of raw QD trajectories (gray) and the drift-corrected QD trajectories (red) obtained by subtracting the drift path. G , Example images of fixed hippocampal neurons expressing the synaptic marker tdTomato-Homer1c (background pixels) and the YFP-GluN1-1a subunit labeled and tracked with the indicated QD-based probes (red). H , Scatter plots of all QD localizations; the red “+” indicates the mean value in both axes. I , Histograms showing the distribution of the distances between each QD localization shown in H ; the data were fitted with a Gaussian function, and the corresponding sigma (σ) values are indicated. J , Box plot summarizing the Gaussian fits performed on fixed QDs ( n = 20/group); one-way ANOVA F (2,57) = 121.84, p < 0.0001 followed by Bonferroni's multiple-comparisons test with p -values denoted in the figure. The average (mean ± SEM) localization errors were σ = 6.62 ± 0.25 nm (antiGFP-QD605), σ = 6.98 ± 0.32 nm (nanoGFP-QD605), and 15.34 ± 0.66 nm (nanoGFP-QD525).

Article Snippet: The bacterial expression vector encoding GFP nanobody (nanoGFP; catalog #49172, Addgene) was a gift from Brett Collins (The Institute for Molecular Bioscience, Queensland).

Techniques: Expressing, Staining, Incubation, Marker, Infection, Labeling

Journal: Nature communications

Article Title: Virus-modified paraspeckle-like condensates are hubs for viral RNA processing and their formation drives genomic instability.

doi: 10.1038/s41467-024-54592-5

Figure Lengend Snippet: Fig. 3 | The viral protein ORF11 associates with v-mPS. A Graph of proteins over a 10% abundance ratio and 4% abundance change in the SFPQ TMT-MS at 24 h (n = 2). ORF11 is highlighted in orange, proteins involved in RNA processing are highlighted purple and DEAD/DEAH box helicases and hnRNP proteins are blue. Proteins con- firmed via IF as co-localising are marked with arrows. B Western blot analysis of FLAG Co-IPs in TREx-BCBL1-RTA FLAG-ORF11 O/E cells during latent or 24 h post- lytic replication and probed with antibodies against FLAG (ORF11), SFPQ, ORF57 and GAPDH (n = 3). C Western blot analysis of FLAG Co-IPs in TREx-BCBL1-RTA

Article Snippet: Protein purification Wildtype ORF11 cloned into popinJ vector (Addgene: #26045) was transformed into Lemo21(DE3) competent cells and innoculated in 1 L of ampicillin selective LB broth at 37 °C with 180 RPM shaking until OD600 reached 0.6 nm absorbance.

Techniques: Western Blot