mouse anti α tubulin  (Novus Biologicals)

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: Ticks encode a single ancestral locus expressing ANP32A variants that support BRBV polymerase activity. ( A ) Syntenic gene blocks were identified for ANP32 genes, revealing a single ancestral locus present in the lone star (Aam) ticks and brown dog (Rsa) ticks. Comparisons were made to humans (Hsa, Homo sapiens ), chickens (Gga), swine (Ssc), and cows (Bta). ( B ) De novo transcriptome assembly identifies multiple splice variants of tick ANP32. The genomic locus for Rsa and Aam ticks is indicated, as well as accession numbers for previously cataloged transcript variants. Exons are shown as boxes with non-coding regions in pink, open reading frames in red, and a small upstream open reading frame (uORF) in gray. The conserved start site present in vertebrate ANP32A is indicated. ( C ) BRBV polymerase activity assays were performed in DKO cells expressing the viral polymerase, nucleoprotein, and a viral reporter as well as the indicated ANP32A or ANP32B from humans (hu), cows (co), chickens (ch), lone star ticks (Aam), or brown dog ticks (Rsa). Naming of tick variants corresponds to those diagrammed in (B). Activity was normalized to an internal Renilla luciferase control. ( D ) Polymerase activity assays were performed for DHOV and THOV as in (C). ( E ) Activity of the FLUAV polymerase in cells expressing diverse ANP32 proteins was measured as described for (C). For (C)–(E), data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against the empty vector. ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant. ( F ) Expression of ANP32 proteins was detected by western blot. Tubulin was probed as a loading control.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Expressing, Activity Assay, Luciferase, Control, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: BRBV polymerase activity is sensitive to changes at the N-terminus of ANP32. ( A ) Alignment of the N-terminus of human ANP32A (hu32A), ANP32B (hu32B), and lone star tick (Aam) ANP32A (Aam 32). Mutants used below are highlighted in red. ( B–D ) Changes to the N-terminus of ANP32 disrupt its ability to support BRBV polymerase. ( B–G ) Polymerase activity assays for the indicated polymerase were performed in DKO cells expressing huANP32A with a C-terminal FLAG tag (C-FLAG), an N-terminal FLAG tag (N-FLAG), a C-terminal FLAG tag with an N-terminal deletion (∆ME) or mutation (E2A), or an empty vector control. Polymerase lacking the PB2 subunit (∆PB2) served as a negative control. ( H–I ) Polymerase activity assays were performed with the indicated polymerase in DKO cells expressing huANP32B with a C-terminal FLAG tag (C-FLAG), an N-terminal FLAG tag (N-FLAG), a C-terminal FLAG tag with an N-terminal deletion (∆MD) or mutation (D2A), or an empty vector control. Polymerase lacking the PB2 subunit (∆PB2) served as a negative control. For all, polymerase activity was normalized to an internal Renilla luciferase control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against huANP32 C-FLAG. **** P ≤ 0.0001; ns, not significant. ( J ) Representative blot of ANP32 proteins and variants confirming equivalent expression. Tubulin was probed as a loading control.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Expressing, FLAG-tag, Mutagenesis, Plasmid Preparation, Control, Negative Control, Luciferase

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: The impact of adaptive variants in chicken ANP32A and ANP32B differs between BRBV and FLUAV polymerases. ( A–D ) Polymerase activity assays were performed in DKO expressing the indicated viral polymerase, nucleoprotein, and a viral reporter. Cells were complemented with human (huANP32), chicken (chANP32), chANP32A lacking residues 175-207 (ch∆33), or mutant huANP32B N129I/D130N proteins. Activity was normalized to an internal Renilla luciferase control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons. ANP32A variants were compared against huANP32A, and ANP32B variants were compared against huANP32B. * P ≤ 0.05; *** P ≤ 0.001; ns, not significant. ( E ) Representative blot confirming expression of ANP32 variants. Tubulin was detected as a loading control.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Expressing, Mutagenesis, Luciferase, Control

Journal: Journal of Virology

Article Title: ANP32 proteins from ticks and vertebrates are key host factors for replication of Bourbon virus across species

doi: 10.1128/jvi.00522-25

Figure Lengend Snippet: Amino acid variations in the N-terminus and central regions of ANP32E preclude its use by BRBV or FLUAV, respectively. ( A ) Diagram of chimeric proteins between human ANP32A and ANP32E. Fusions were made after residues 120 and 170. ( B ) BRBV polymerase, nucleoprotein, and a viral reporter were expressed in DKO cells with the indicated human ANP32A, ANP32E, or chimeric clone. Activity was normalized to an internal Renilla luciferase control. ( C ) FLUAV polymerase activity was measured as in (B). ( D ) Representative blot showing expression of ANP32 proteins and chimeras. Tubulin was detected as a loading control. Data are mean of n = 3 ± sd. Significance was assessed by a one-way ANOVA with a post hoc Dunnett’s multiple comparisons test against human ANP32A or ANP32E. *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Article Snippet: Primary antibodies used were mouse α-tubulin DM1A (Proteintech 66031-1-Ig), α-actin (Proteintech 66009-1-Ig), α-human ANP32A (Proteintech 67687-1-Ig), α-human ANP32B (Proteintech 10843-1-AP), α-V5 (Chromotek v5ab), and M2 α-FLAG (Sigma F1804).

Techniques: Activity Assay, Luciferase, Control, Expressing

Journal: iScience

Article Title: Actin fenestrae amplify the membrane response to hypertonic stress in osmosensory neurons

doi: 10.1016/j.isci.2025.112042

Figure Lengend Snippet:

Article Snippet: Anti α-tubulin mouse monoclonal antibody (DM1A, Millipore Sigma, 1:500).

Techniques: Recombinant, Membrane, Blocking Assay, Proximity Ligation Assay, In Situ, Software

Journal: The Journal of Clinical Investigation

Article Title: Neonatal but not juvenile gene therapy reduces seizures and prolongs lifespan in SCN1B –Dravet syndrome mice

doi: 10.1172/JCI182584

Figure Lengend Snippet: ( A ) Absence of Scn1b mRNA in null somatosensory cortex. **** P < 0.0001. ( B – I ) mRNA abundance in P16–18 mouse somatosensory cortex; RT-qPCR data normalized to WT. Data were analyzed using 2-tailed, unpaired t test except in E , analyzed by 1-way ANOVA. ( B ) Scn1b in untreated and AAV-Navβ1–treated null. *** P < 0.005. ( C ) Scn1b in AAV-Navβ1–treated P16–18 WT and null. * P < 0.05. ( D ) Scn1b in untreated and AAV-Navβ1–treated WT. P = 0.1225. ( E ) Scn1b in untreated and AAV-Navβ1–treated WT and null. P = 0.1003. ( F ) Scn1a in untreated WT and null. **** P < 0.0001. ( G ) Scn1a in untreated and AAV-Navβ1–treated null. ** P < 0.0074. ( H ) Scn1a in AAV-Navβ1–treated WT and null. P = 0.4626. ( I ) Scn1a in untreated versus AAV-Navβ1–treated WT. P = 0.9431. ( J ) Western blot analysis of WT (+/+) or null (–/–) mouse brain. Immunoblot (IB): Anti-β1. Bottom: IB of the same blot with anti-tubulin. ( K ) Western blot analysis of AAV-Navβ1–treated WT (+/+) or null (–/–) brains with or without PNGase F treatment. IB: Anti-β1. Arrow, deglycosylated β1 immunoreactive bands. ( L ) Single i.c.v. dose of AAV-Navβ1 at P2 improved survival of null (light blue) versus untreated (purple) or AAV-EV–treated null mice (black) through P160 ( P < 0.0001, log-rank Mantel-Cox test). Dark blue, untreated WT mice. Kaplan-Meier (Wilcoxon) analysis. ( M ) Single i.c.v. dose of AAV-Navβ1 at P2 did not increase body weights of null (solid blue line) versus WT mice (dashed black line) through P80 (**** P < 0.0001, 2-way ANOVA). ( N ) Single i.c.v. dose of AAV-Navβ1 at P10 had no effect on null (black) versus untreated null mouse survival (purple; data taken from L ) (log-rank Mantel-Cox test). ( O ) Single i.c.v. dose of AAV-Navβ1 at P2 prevented hyperthermia-induced seizures in P16 null mice. Kaplan-Meier analysis presented for first observed Racine scale 5–6 seizure; uninjected null (purple, n = 5), uninjected WT (dark blue, n = 5), AAV-Navβ1–injected null (light blue, n = 6), or AAV-Navβ1–injected WT mice (black, n = 5). **** P < 0.0001.

Article Snippet: Anti–α-tubulin antibody (1:1,000; Cedarlane CLX135AP) was used to control for equal sample loading.

Techniques: Quantitative RT-PCR, Western Blot, Injection