Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) A schematic diagram of the RNA immunoprecipitation (RIP) assay used to detect METTL3-associated N mRNA. (B) 293T cells were transfected with METTL3 expression plasmids. At 24 h post-transfection (hpt), the cells were infected with rBPIV3-EGFP at an MOI of 1 for 48 h. The infected cells were lysed, and the extracts were subjected to RNA immunoprecipitation assay with anti-FLAG antibody (Ab). After immunoprecipitation, cDNA of BPIV3-N mRNA captured by METTL3 was synthesized and the amount of N mRNA was quantified by qPCR. Followed by normalization to input RNA values, relative enrichment values of N mRNA were expressed relative to control IgG values using the ΔΔCt method. (C) SRAMP was used to predict the m6A site of the N gene derived from the BPIV3 genome. The vertical axis shows the m6A score and the horizontal axis shows the position number of the base of the N gene where N6-methyladenosine is present within the m6A motif. A yellow bar indicates low-scoring m6A site, N327, and red bars indicate high-scoring m6A sites, N872, N1146, N1372, N1427, and N1443. (D) Schematic representation of the MeRIP (m6A RNA immunoprecipitation) assay. (i) The m6A-containing RNA fragments were bound by an m6A-specific Ab. (ii) Viral N mRNA containing m6A modifications was fragmented. (iii) Antibody–RNA complexes were captured using protein affinity beads. (iv) RNA was eluted, reverse transcribed into cDNA, and amplified by qPCR using primers specific for the indicated N gene regions (N327, N872, N1146, and N1372–1443). (E) 293T cells were infected with rBPIV3-EGFP at an MOI of 1. At 72 h post-infection, the infected cells were harvested, and only mRNA from total RNA was purified. The m6A-modified mRNA was immunoprecipitated with m6A-specific Ab and magnetic beads. Followed by cleavage of the captured mRNA with the cleavage enzyme, the m6A-specific Ab-captured mRNA was eluted from magnetic beads and purified. cDNA was synthesized by reverse transcription reaction, and the m6A sites in N mRNA were then identified by qPCR. The relative value of each to the mRNA value of input RNA was calculated using the ΔΔCt method. Data represent the mean ± SD from n = 3 independent experiments. Asterisks represent statistically significant differences (* p < 0.05). ns; not significant.

Article Snippet: For dsRNA detection, mouse anti-dsRNA Ab was directly labeled using a FlexAble mouse IgG2a labeling kit (Proteintech).

Techniques: RNA Immunoprecipitation, Transfection, Expressing, Infection, Immunoprecipitation, Synthesized, Control, Derivative Assay, Reverse Transcription, Amplification, Purification, Modification, Magnetic Beads

Journal: The Journal of Cell Biology

Article Title: Endoplasmic reticulum patterns insect cuticle nanostructure

doi: 10.1083/jcb.202503127

Figure Lengend Snippet: The role of Ref(2)P in Gox localization and ubiquitination. (A) Ref(2)P is specifically enriched in the shaft region of olf hair cells, which is labeled by HA–Gox. Maxillary palp 42 h APF. (B) Subcortical localization of Ref(2) in control and gox mutant olf hair cells, and their quantification. Line intensity profiles were obtained from 5–7 individual hairs per genotype using the PlotProfile function in Fiji and averaged after normalization with custom Python scripts. Gray line: Ref(2)P distribution in control; magenta line: Ref(2)P in gox KO. Error shading represents mean ± SD. (C) Subcortical Gox localization was lost in Ref(2)P RNAi. Right graph: quantification. Right panel: quantification of fluorescence intensity profiles. Gray line: HA–Gox in control; magenta line: HA–Gox in Ref(2)P knockdown cells. No formal statistical test was applied because the plots represent averaged fluorescence intensity distributions (D) Interaction of Gox2 with Ref(2)P and ubiquitination in S2 cells. Gox–Flag and Ref(2)P-Myc were co-expressed and immunoprecipitated with the tag antibodies. Immunoprecipitate of Ref(2)P containing a low molecular weight form of Gox (*, lane 4). The increased amount of high molecular weight forms of Gox was induced by Ref(2)P (compare lane 7 and 8). Note that cross-reactivity of HRP-conjugated antibodies to the IgG (marked with **) used for immunoprecipitation. (E) Properties of Gox8KR. Gox8KR (*) and Ref(2)P (**) were co-immunoprecipitated with each other (lane 8 and 12). *** indicates the band of Ref(2). Although Ref(2) caused a slight increase in Gox amount and molecular mass (lane 16), no specific increase in ubiquitination level was observed (lane 20). (F) WT gox and gox8KR were expressed by neur-Gal4. (G) Line intensity profiles showing the distribution of Flag (pink) and ER marker tdTomato–Sec61b (gray) in olf hair cells expressing UAS–Gox–Flag (top) or UAS–Gox8KR–Flag (bottom). Profiles were obtained and averaged as in B and C. No formal statistical test was applied; error shading represents mean ± SD. (H) AlphaFold 2 model of Gox/Osi23 (pLFFT score 68.12), and the location of the two mapped ubiquitinated lysine (K101 and K131) identified by the LC-MS/MS. Alpha helices are labeled (H1–H6). Molecular weight markers (kDa) are indicated for all blots, and uncropped images are provided in SourceData FS4.pdf.

Article Snippet: The FlexAble CoraLite Plus 488 Antibody Labeling Kit for Mouse IgG1 was used to label the mouse anti-Cnx antibody for double labeling with two mouse antibodies.

Techniques: Ubiquitin Proteomics, Labeling, Control, Mutagenesis, Fluorescence, Knockdown, Immunoprecipitation, Molecular Weight, High Molecular Weight, Marker, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific reports

Article Title: Metabolic phenotype of bovine blood-derived neutrophils is altered in milk.

doi: 10.1038/s41598-025-93929-y

Figure Lengend Snippet: Fig. 1. Characterization of immune cells in blood (a–e) and milk samples from healthy (f–j) and inflamed udder quarters (k–o). Representative cytospin preparations of blood cells (a) and milk cells from healthy (f) and inflamed (k) udder quarters in Haema Quick staining visualises different cell populations and morphology. Representative SSC/FSC density plots of viable CD45+ blood leukocytes (b) and viable CD45+ milk leukocytes from healthy (g) and inflamed (l) udder quarters demonstrate divergent leukocyte subpopulations. Representative dot plots of CD4/CD8 (c,h,m), CD172a/CD14 (d,i,n), and CD45/CH138A (e,j,o) staining show differences within the leukocyte subpopulations between blood cells (c–e) and milk cells from healthy (h–j) and inflamed (m–o) udder quarters in more detail.

Article Snippet: The following primary antibody combinations were used: mouse anti-bovine CD4 (isotype IgG2a, clone ILA11A, Kingfisher, Saint Paul, MN, USA; 1:400) labeled with FlexAble CoraLite ® Plus 555 antibody labeling kit for mouse IgG2a (ChromoTek & Proteintech Germany, Planegg-Martinsried, Germany) and mouse anti-bovine CD8 (isotype IgG1, clone CC63, Bio-Rad; 1:200), mouse anti-bovine CD172a (isotype IgG1, clone DH59B, Bio-Rad; 1:10) and biotin-conjugated mouse anti-human CD14, cross-reactive to bovine (isotype IgG2a, clone TÜK4, Bio-Rad; 1:100), mouse antibovine granulocytes (isotype IgM, clone CH138A, Kingfisher; 1:200) and mouse anti-equine MHC class II, cross-reactive to bovine (isotype IgG2a, in house antibody; 1:400), mouse anti-bovine CD25 (isotype IgG1, clone IL-A111, Bio-Rad; 1:200) or mouse anti-human CD62L, cross-reactive to bovine (isotype IgG2b, clone FMC46, Bio-Rad, 1:50).

Techniques: Staining