rabbit anti mxa polyclonal antibody pab  (Proteintech)

Journal: bioRxiv

Article Title: Host-specific bacterial modulation of airway gene expression and alternative splicing

doi: 10.1101/2025.07.18.665426

Figure Lengend Snippet: A) Dotplot of epithelial barrier gene lists where each row is a different gene list and each column a different sample. Samples are annotated by color for their microbial treatment, donor, and time point. Points are colored based on their gene score (median log 2 fold change (FC) over vehicle control). B) Heatmap of transcriptional response in a manually curated list of antibacterial innate immunity (non-interferon cytokines and antimicrobial peptides). Each row represents a different gene and each column a different sample. Columns were hierarchically clustered. Each column was annotated by color with the sample’s microbial treatment, donor cells, and time point. Cells were colored by the log 2 FC relative to the appropriate vehicle control. Outlined boxes have an adjusted P-value < 0.05. Gray boxes represent genes that were filtered out due to low gene counts at a specific time point. Genes that were not present in at least 2/3 time points were removed. C) Dotplot of the antibacterial score for each sample. Points are colored by donor according to the legend in . Error bars represent the 95 th confidence interval determined by bootstrapping. D) Heatmap of interferon stimulated genes (ISGs) relative to vehicle control as in . E) Dotplot of the ISG score for each sample as in . F) Representative immunofluorescence images of ISG MX1 (red) and DAPI/nuclei (blue) following 48 hours of colonization. n = 3 ALI cultures. G) Dotplot of the log 2 FC for PIAS3 expression at 12 hours. Each point represents a different microbial treatment, and points were colored yellow if that microbe-donor pairing resulted in ISG expression at 24 or 48 hours. H) Boxplot of the log 2 fold changes for donors 3, 5, and 6. Wilcoxon test was used to compare between ISG stimulators and non-stimulators. For relevant plots, * represented P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, and ns = non-significant. For boxplots, box middle represents the median, box edges represent 25 th and 75 th quartiles, and outlier values are separate points.

Article Snippet: The sections were stained with either anti-MX1 primary antibody (polyclonal Rabbit N2C2, #GTX110256, GeneTex) or anti-SCGBA1 (Clone 394324, R&D) and anti-acetylated-alpha-tubulin (Clone 6-11B-1, Thermofisher) for one hour followed by the appropriate secondary antibody for 30 min in 1X PBS/5% BSA/0.05% saponin.

Techniques: Control, Immunofluorescence, Expressing

Journal: BMC Biology

Article Title: Species- and strain-specific microbial modulation of interferon, innate immunity, and epithelial barrier in 2D air–liquid interface respiratory epithelial cultures

doi: 10.1186/s12915-025-02129-7

Figure Lengend Snippet: Species and strain level differences in microbial induction of antiviral interferon pathways. A Heatmap (log 2 fold change (FC)) of the transcriptional response of interferon-stimulated genes (ISGs) in ALIs colonized with microbes for 48 h relative to vehicle control. Each column is a microbial treatment and each row is an ISG. Outlined cells indicate FDR adjusted P -value < 0.05. Microbial treatments were classified as interferon non-stimulator and stimulator based on hierarchical clustering. ISG score was determined based on the median log 2 FC of the ISGs. Below the heatmap was each microbial treatment’s ISG score. B Boxplot of ISG scores of ISG non-stimulator (purple) vs. stimulator (green). Each point represented a microbial treatment; box edges indicated 25th and 75th percentiles. C Dot plot of each microbe’s ISG score where microbial treatments were sorted by genus. Dots were colored based on interferon category. D Bar graph of the log 2 FC for each ISG for each Rothia dentocariosa strain, one of which was an interferon non-stimulator, the other a stimulator. E Volcano plots of DEGs for the three microbial treatments used for immunofluorescence validation. Genes (points) colored in blue were significantly downregulated (FDR adjusted P -value < 0.05), pink were significantly upregulated, and red were significantly upregulated ISGs. F , G Immunofluorescence of ALI colonized for 48 h with a microbe or vehicle ( n = 3 ALI from single experiment). F Quantification of MX1 by histocytometry with the percentages of DAPI + MX1 + cells per condition. Data shown were representative of 3 technical replicates per condition with at least 2 ALI sections per replicate. G Representative immunofluorescence of ALI after 48 h of bacterial/vehicle exposure stained for nuclei (DAPI), and MX1 (red) to reveal downstream of IFN response. Scale bar 50 μm, in white on the left corner. For relevant plots, statistical analysis was two-sided Mann–Whitney U test with Bonferroni correction. For all relevant plots, * indicated P -value < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Article Snippet: The sections were then stained with anti-MX1 primary antibody (polyclonal Rabbit N2C2, #GTX110256, GeneTex, Irvine, CA, USA) for 1 h followed by secondary antibody (anti-rabbit IgG AF555, #406,412, BioLegend, San Diego, CA, USA) for 30 min at room temperature in 1X PBS/5% BSA/0.05% saponin.

Techniques: Control, Immunofluorescence, Biomarker Discovery, Staining, MANN-WHITNEY

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , PacBio HiFi sequencing improves genome assembly contiguity. UCSC genome browser screenshot showing a 700 kb locus of the human chr20 and genome alignments to six rhinolophid bats (boxes in the alignment net represent aligning sequence and connecting lines deletions or unaligning sequence). The region highlighted in grey does not align between human and Rhinolophus ferrumequinum , because the R. ferrumequinum PacBio CLR-based assembly has a large 421,369 bp assembly gap in this locus. As a result, several genes contained in this locus are missing from this assembly, which contributes to the slightly higher number of missing genes for R. ferrumequinum vs. other rhinolophid bats (Fig. ). Consistent with PacBio HiFi-based assemblies often having higher contig N50 and N90 values (Fig. ), the other four HiFi-based rhinolophid assemblies have a contiguous sequence in this locus without an assembly gap. Rhinolophus sinicus , which was assembled from Illumina short reads, has 24 smaller assembly gaps with sizes 15–1058 bp in this locus. b-f , Lower assembly quality leads to missed signals of gene selection. To explore the effect of assembly quality on results from genome-wide selection screens, we replaced the high-quality Bat1K assembly (HLrhiFer5) of the Greater horseshoe bat ( Rhinolophus ferrumequinum ) with a previous short read based assembly (rhiFer1) of the same species that has a higher degree of incompleteness and fragmentation (panel b-d). We kept all other 114 mammalian species and tested if the genes under selection in the R. ferrumequinum branch with the HLrhiFer5 assembly are also under selection when using the rhiFer1 assembly. Similarly, we replaced the Bat1K assembly (HLrouAeg4) of the Egyptian rousette ( Rousettus aegyptiacus ) with a previous assembly (Raegyp2.0) that used long and short read data but has a smaller contig N50 and an excess of inactivating mutations (indicative of a higher base error rate; see Supplementary Fig. ). We identified 272 vs. 133 genes under positive selection in HLrhiFer5 vs. rhiFer1, and 299 vs. 194 genes under positive selection in HLrouAeg4 vs. Raegyp2.0, indicating that lower assembly quality hampers the identification of selected genes. To illustrate this, the panels show UCSC genome browser screenshots of five immune-related genes, where we detected positive selection in the Bat1K but not the previous assembly. The first three examples (b-d) show cases where the previous assembly does not cover the gene on a single scaffold and exons are missing because of assembly gaps. The last two examples (e-f) show how assembly problems other than assembly gaps hamper selection screens. b , LAT2 (linker for activation of T cells family member 2), a regulator of T cell activation , is split across two different scaffolds in rhiFer1, as shown by the two alignment chains between human (hg38 assembly) and R. ferrumequinum rhiFer1. Importantly, while other methods can capture only one of these gene fragments at best, TOGA recognizes both alignment chains as orthologous and joins both gene fragments, resulting in a more complete codon alignment. Nevertheless, coding exon 8 (blue highlight) is missing in rhiFer1, thus this exon is missing in the codon alignment. In HLrhiFer5, all exons align to a single scaffold. c , PPP6C (protein phosphatase 6 catalytic subunit), a factor that regulates STING phosphorylation and activation , is split across two scaffolds (alignment chains) in rhiFer1. While TOGA recognizes the red chain as an orthologous fragment of PPP6C , the inset shows that coding exon 1 overlaps an assembly gap in rhiFer1 (but not HLrhiFer5), thus this exon will be missed in the codon alignment. d , The gene locus of FOXP3 (forkhead box P3), a master regulator involved in regulatory T-cell development and function , is split across several scaffolds in rhiFer1. Although the entire coding region is present on a single scaffold (brown chain), the last coding exon overlaps an assembly gap in rhiFer1 and thus will be missed in the codon alignment. e , CD48 (CD48 antigen), a cell surface factor involved in adhesion and activation of adaptive immune cells , lacks an aligning exon 1 in the Raegyp2.0 assembly. Compared to the HLrouAeg4 assembly, Raegyp2.0 lacks ~22,800 bp of sequence and this sequence is also present in other Pteropodid assemblies. This indicates that this ‘deletion’ is likely an assembly error in Raegyp2.0. f , MX1 (MX dynamin like GTPase 1), an interferon-induced antiviral gene , has two orthologous alignment chains that cover the gene. While this apparent ‘duplication’ is likely due to incomplete haplotype purging in Raegyp2.0, it leads TOGA to classify MX1 as a 1:2 ortholog in this assembly and since our screen only considers 1:1 orthologs, this gene is missed in a screen including Raegyp2.0.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Sequencing, Selection, Genome Wide, Activation Assay, Phospho-proteomics

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , ISG15 Cys mutants do not alter ISG expression during H1N1 PR8 Influenza A virus (IAV) infection or ISG15 supernatant treatment in epithelial cells. To investigate why mutating or deleting Cys78 from human ISG15 and restoring Cys78 in R. affinis ISG15 significantly increased IAV production, we tested whether the ISG15 Cys mutants but not wildtype ISG15 impacted IRF3 activation (known to be modulated by ISG15 ), IFN production, and induction of key antiviral interferon-stimulated genes (MX1 and IFIT1). IAV was infected at an MOI of 0.1 for 48 h after transfection with wildtype ISG15 constructs and ISG15 Cys78 mutants (indicated on right). The representative western blot shows that IAV infection causes some IRF3 activation and only minimal MX1 or IFIT1 protein induction in Vero-E6 cells when transfected with an empty vector or most ISG15 constructs. While R. affinis S77C induced more MX1 and IFIT1, this did not alter IAV production (Fig. ), indicating minimal effect on the final production of infectious IAV particles. This is consistent with VeroE6 cells having limited IFN signal amplification, and shows that while ISG15 modulates IFN signaling and some ISG-induction in Vero-E6 cells, other effects of ISG15 on viral production are more relevant. Importantly, there is no impact of Cys-removal from human ISG15 on MX1 or IFIT1 in infected cells, indicating that the observed pro-viral outcome of ISG15 Cys mutants is likely not due to downregulating interferon-stimulated gene expression. A representative western blot image is shown from three independent experiments. b , Extracellular ISG15 does not enhance NFκB and IFN signaling in A549 cells. Wildtype and Cys-mutant ISG15 showed limited anti-IAV activity. To rule out a reliance on extracellular cytokine enhancement from ISG15, we measured the effect of extracellular wildtype or mutant ISG15 on NFκB and IFN signaling. As this process requires the ISG15 receptor, LFA-1 , which is not expressed in VeroE6 cells, we used A549 cells that are IFNɣ-competent and express a low-level of LFA-1. ISG15-containing supernatants from IAV-infected VeroE6 cells were UV-treated with 10000 gy of UV-C to deactivate IAV. A549 cells were then treated with these supernatants overnight. We then measured A549-cell lysates for TNFα and IL-6 to assess NFκB-signaling, and IFIT1, IFITM3, and endogenous ISG15 to assess IFN signaling. We observed no obvious induction of these pathways in these cells between wildtype and Cys-mutant ISG15, indicating the pro-viral effect of ISG15 mutants is likely not caused by impairing ISG15’s cytokine enhancement function in these cells. A representative western blot image is shown from three independent experiments.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Expressing, Virus, Infection, Activation Assay, Transfection, Construct, Western Blot, Plasmid Preparation, Amplification, Gene Expression, Mutagenesis, Activity Assay