Journal: The Journal of Biological Chemistry

Article Title: A biallelic variant of the RNA exosome gene, EXOSC4 , associated with neurodevelopmental defects impairs RNA exosome function and translation

doi: 10.1016/j.jbc.2024.107571

Figure Lengend Snippet: The pathogenic amino acid substitution in EXOSC4 decreases the steady-state level of the protein and can alter interactions with other RNA exosome subunits. A , the murine EXOSC4-L187P variant, corresponding to the human EXOSC4 variant identified in patients, is present at a lower steady-state level than WT murine EXOSC4 in a mouse neuronal cell line. Lysates of mouse N2a cells transfected with empty vector or vector expressing murine Myc-EXOSC4 or Myc-EXOSC4-L187P were analyzed by immunoblotting with anti-Myc antibody to detect Myc-EXOSC4 proteins. The stain-free signal serves as a loading control and neomycin phosphotransferase II (NPTII) serves as a transfection control. B , quantitation of the relative level of EXOSC4-L187P protein compared to EXOSC4 detected in the lysates of N2a cells expressing Myc-tagged EXOSC4 or EXOSC4-L187P from four immunoblot experiments – one shown in ( A ). The graph shows the relative level of EXOSC4-Myc protein compared to WT EXOSC4 (WT) from four independent experiments (n = 4). Error bars represent SEM. Statistical significance is calculated by a Student’s t test (∗∗ p -value ≤ 0.01). C , Myc-EXOSC4 or Myc-EXOSC4-L187P was immunoprecipitated from N2a cells and interactions with the RNA exosome subunits EXOSC8, EXOSC9, and the RNA exosome–associated EXOSC10 were analyzed by immunoblotting. Both the input and bound samples are shown for Myc-EXOSC4 and Myc-EXOSC4-L187P. NPTII represents the neomycin phosphotransferase II, which is encoded on the Myc-EXOSC4/EXOSC4-L187P plasmids, indicating similar levels of transfection for EXOSC4 and EXOSC4-L187P. Hsp90 serves as a loading control.

Article Snippet: For transfection control, Neomycin phosphotransferase II (NPTII) expressed from NeoR cassette on pcDNA3 -Exosc4 vectors was detected with mouse anti-NPTII monoclonal antibody (1:1000; Cell Applications, Inc.; Cat. CP10330).

Techniques: Variant Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Staining, Control, Quantitation Assay, Immunoprecipitation

Journal: Journal of neurochemistry

Article Title: Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

doi: 10.1111/jnc.13552

Figure Lengend Snippet: Fig. 1 (a) Gpm6a and Rac1 co-immunoprecipitate with Coro1a from rat hippocampal lysates. Western blot of proteins co-immunoprecipi- tated from rat hippocampal lysates using anti-Coro1a antibody and probed with anti-Gpm6a, anti-Rac1 and anti-Coro1a antibodies. For this purpose, the membrane (different kDa areas) was cut and incubated with indicated antibodies. Non-immune rabbit serum was used as a control. Gpm6a and Rac1 are present in the anti-Coro1a immunoprecipitate. Bands representing Gpm6a are indicated by stars. (b) Coro1a co-localizes with Gpm6a in hippocampal neurons. Confocal image of hippocampal neurons (4 DIV) co-immunostained with anti- bodies against Gpm6a (red), Coro1a (green) and dendritic marker MAP2 (blue). A portion of Gpm6a-labeled spots co-localizes with Coro1a (arrowheads; insets 1 and 2). Scale bar, 20 lm.

Article Snippet: Mammalian expression plasmids: pRFP-C1 encoding the red fluorescent protein (RFP), pEGFP-C1 (Clontech Laboratories, © 2016 International Society for Neurochemistry, J. Neurochem. (2016) 137, 46--61 Mountain View, CA, USA) encoding the enhanced green fluorescent protein (EGFP), RFP-tagged wild-type (wt) Gpm6a (Gpm6aRFP) described previously (Alfonso et al. 2005), EGFP-tagged wt Coro1a (wtCoro1a-EGFP), and a deletion mutant containing only 5 WD repeats (aa 65-306) of Coro1a [Coro1a(WD1-5)-EGFP] kindly provided by Dr William Trimble (Yan et al. 2005), EGFPtagged Rac1 T17N [Rac1DN-EGFP; Addgene#12982 (Subauste et al. 2000)], EGFP-tagged Rac1 Q61L [Rac1CA-EGFP; Addgene#12968 (Subauste et al. 2000)], myc-tagged Pak1 K299R [Addgene#12210 (Sells et al. 1997)], and myc-tagged Pak1 H83L/H86L [Addgene#12211 (Sells et al. 1997)]. siRNAs: siGENOME non-targeting siRNA Pool #2, Coro1a siRNAs targeting either its coding DNA sequence (CDS) [prevalidated in Suo et al. (Suo et al. 2014)] or its 30-UTR region (custom designed).

Techniques: Western Blot, Membrane, Incubation, Control, Marker, Labeling