tap73  (Novus Biologicals)

Journal: iScience

Article Title: Targeting of Itch by clomipramine or gene therapy improves cognitive defects related to Alzheimer’s disease

doi: 10.1016/j.isci.2026.115181

Figure Lengend Snippet: Clomipramine inhibits Itch and prevents neuronal apoptosis and cell cycle re-entry in neurons from TgAD mice (A) Chemical structure of clomipramine. The chloride moiety is proposed to interact with catalytic cysteine in the HECT domain of Itch. (B) Rat cortical neurons were treated with Aβ 42 and/or clomipramine (75 nM) for 48 h followed by 12 h treatment with the proteasome inhibitor MG132. TAp73 was immunoprecipitated, and western blotting was performed on TAp73-IP with anti-ubiquitin and anti-Itch antibodies. Total protein lysates were also used for western blotting with indicated antibodies. Ubiquitinated TAp73 levels were quantified by densitometry of ubiquitin immunoblot, normalized with respect to the input TAp73, which was also normalized with respect to actin (mean ± SEM, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA, Tukey’s test, N = 3). (C) Rat cortical neurons were treated with Aβ 42 and/or 75 nM clomipramine for 48 h followed by western blotting for indicated proteins. The levels of TAp73, PCNA, and cl_caspase3 were quantified by densitometry, and fold change with respect to untreated (ctrl) neurons was determined (mean ± SEM, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA, Tukey’s test, N = 3). (D) Cortical neurons from WT or TgAD mice were treated with 75 nM clomipramine for 48 h followed by western blotting for indicated proteins. TAp73, PCNA, and cl_caspase3 levels were quantified by densitometry, and fold change with respect to untreated WT neurons is provided (mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA, Tukey’s test, N = 2).

Article Snippet: PCNA (Santa cruz, cat. no. sc-56; 1:500 for WB, 1:50 for IHC), cleaved caspase 3 (CST, cat. no. 966l; 1:1000 for WB), Itch (CST, cat. no. 12117; 1:1000 for WB), TAp73 (Novus, cat. no. NBP2-24737, 1:1000 for WB, 1:50 for IP), myc-tag (9B11) (CST, cat. no. 2276; 1:2000 for WB, 1:100 for IHC), NeuN (Novus, cat. no. NBP2-67314; 1:100 for IHC), Ubiquitin (Santa cruz, cat. no. sc-8017; 1:1000 for WB) and β-Actin (Santa cruz, cat. no. sc-47778, 1:2000 for WB).

Techniques: Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: FDXR Upregulation by p63/p73 is a Prognostic and Therapeutic Marker of HTLV-1-Associated Adult T Cell Leukemia/Lymphoma

doi: 10.64898/2026.02.25.707207

Figure Lengend Snippet: (A,B) RNA-Seq data retrieved from Protein Atlas were stratified by FDXR expression (≥75 nTPM = Mid-high_FDXR, <75 nTPM = Mid-low_FDXR). Differential transcription factor (TF) activity between groups was inferred using VIPER , based on regulatory networks constructed with DoRothEA and ARACNe. The bubble plot in (B) ranks TFs based on their differential activity in the two groups. The colour of the dots reflects relative TF activity, while the size of the dots represent -Log10 adjusted p values (FDR) calculated with the limma package. (C–F) Expression of TAp63 mRNA (C), p63α protein (D), TAp73 mRNA (E) and p73 protein (F) in HTLV-1 + , ATL, and uninfected cell lines and primary cells. Relative mRNA expression was quantified by qPCR; protein expression was assessed by western blot. In panels (C,E), each dot represents an independent qPCR assay (n = 3), and data are presented as mean ± SD. Relative mRNA levels were calculated by ΔΔCq using TBP expression as reference. Data were analyzed by non-parametric Wilcoxon rank-sum test. (G,H) Relative expression of TAp63 (G) and TAp73 (H) mRNA in primary CD4 T cells from PLHTLV stratified by disease condition and healthy controls. Relative mRNA expression was quantified by qPCR using the ΔΔCq method and TBP expression as reference. Each dot represents a donor and data are depicted as median ± IQR. Data were analyzed by non-parametric Kruskal–Wallis test followed by Dunn’s post hoc test with Benjamini–Hochberg correction for multiple comparisons. (I,N) Effect of TP63 and TP73 knockout on FDXR expression. The HTLV-1–infected MT-4 cell line (I-K) and the ATL-derived ATL-55T cell line (L-N) were stably transfected with Cas9 and subsequently transduced with lentiviruses expressing either a non-targeting (NT) sgRNA or sgRNAs targeting TP63 or TP73. Stable knockout cell lines were lysed and used to assess protein expression by Western blot. Panels I, L show p63 and FDXR expression in MT-4 and ATL-55T cells, respectively. Panels J, M show p73 and FDXR expression in MT-4 and ATL-55T cells, respectively. Wild-type and non-transduced Cas9-expressing cells served as additional controls. Panels K, N show relative FDXR protein levels normalized using actin as a loading control and the NT sgRNA condition as reference level. Barplots in K, N show mean ± SD for each condition. N = 2. **** p <0.0001; ** p <0.01; *p <0.05.

Article Snippet: The primary antibodies used included anti-FDXR (1:1000, Rabbit, Proteintech #15584-1-AP), anti-p53 (Rabbit, Cell Signaling #2527), anti-p63α (1:1000, Rabbit, Cell Signaling #13109), anti-p73 (1:1000, Rabbit, Cell Signalling #14620), anti-S. pyogenes-Cas9 (1:1000, Mouse, Cell Signalling #14697) anti-VDAC (1:500, Rabbit, Invitrogen #MA5-33205), anti-Tax (1:250, Mouse, Merck #MABF3063), anti-HTLV-1-p24 (1:250, Mouse, Santa-Cruz #sc-53891), anti-HTLV-1-gp46 (1:250, Mouse #sc-53890), anti-alpha Tubulin (1:15000, Rabbit, GeneTex #GTX112141), anti-beta Actin (1:15000, Mouse, Cell Signaling #3700).

Techniques: RNA Sequencing, Expressing, Activity Assay, Construct, Western Blot, Knock-Out, Infection, Derivative Assay, Stable Transfection, Transfection, Transduction, Control

Journal: bioRxiv

Article Title: Cajal-Retzius fate specification is disrupted by constitutive activation of β-Catenin in hem progenitors

doi: 10.64898/2026.02.09.704731

Figure Lengend Snippet: (A) Ai9 is seen in the cortical hem and its derivatives in an E12.5 control and Lmx1aCre; β-Catenin GOF brain. (B) Reelin staining in the same section as (A). (C) TRP73 staining at E14.5 co-localizes with Ai9 in the control but is undetectable in the β-Catenin GOF brain. (D) Genes enriched in CR cells are downregulated in midline tissue β-Catenin GOF brains at E14.5. (E, F) Reelin, βIII-Tubulin and NEUN staining is seen in Ai9+ cells in control brains (arrowheads). In β-catenin GOF brains there is no detectable Reelin (open arrowheads) (E) but βIII-TUBULIN and NEUN staining is seen in Ai9+ cells. (G, J) UMAPs representing tdTomato+ cells from E14.5 control and β-catenin GOF midline, color-coded by cell type (G); by age (H); by genotype (I); showing the expression of Wnt3a, Reln and tdTomato (J). (H) Heatmap of scaled expression of top 10 differentially expressed genes in control and β-Catenin GOF neurons. (I) Dot plots showing scaled expression levels of CR cell enriched genes in control and β-Catenin GOF neurons. Scatterplot in (D) displays Mean ± SEM. Statistical test(D): Multiple Mann-Whitney Tests; p < 0.0001; *p < 0.05; **p < 0.01; ***p < 0.001; ns if p value > 0.05. For (A-C, E-F), N ≥ 3 brains (biologically independent replicates); for (D) N=6 (control), 7 (GOF) biologically independent replicates. Scale bars: 100 μm (all images in A, B, C, E and F).

Article Snippet: Primary antibodies used: Lef1(rabbit, 1:200, CST catalogue #C12A5), β-CATENIN (Mouse, 1:200, BDbiosciences catalogue #610153), β-CATENIN (Rabbit, 1:50, CST catalogue # 8814), RFP (rabbit, 1:200, Abcam catalogue #ab62341), RFP (Mouse, 1:200, Invitrogen catalogue #MA5-15257), β-III TUBULIN (mouse, 1:100, Promega catalogue #G7128), TRP73 (Rabbit, 1:200, CST catalogue #14620S), REELIN (Mouse, 1:200, Millipore catalogue #MAb5364), NEUN (Rabbit, 1:200, invitrogen catalogue #702022).

Techniques: Control, Staining, Expressing, MANN-WHITNEY, IF-P

Journal: bioRxiv

Article Title: Cajal-Retzius fate specification is disrupted by constitutive activation of β-Catenin in hem progenitors

doi: 10.64898/2026.02.09.704731

Figure Lengend Snippet: (A) UMAPs from representing tdTomato+ cells from control and β-Catenin GOF, color-coded by cell type; by genotype; and by pseudo-differentiation trajectory derived from Monocle3. (B) Normalized mRNA expression of Pax6, Eomes/Tbr1, Neurog2, Neurod2, Dcx and Tbr1 across the pseudo-differentiation axis for control (blue) and β-Catenin GOF (red). Thick lines represent Loess smoothed curves. (C) TBR2 staining is seen in Ai9+ cells in controls (arrowheads) but not in β-Catenin GOF brains. Dashed lines mark the ROIs in which TBR2+Ai9+ cells were quantified. (D) The Eomes and Foxj1 expression trajectory along the pseudo-differentiation axis in control neurons (E) Ai9 reporter expression at E12.5 comparing Lmx1a Cre and Foxj1 Cre activity. Foxj1 Cre is not active in hem progenitors but is seen in CR cells and the choroid plexus epithelium. (F) Genes enriched in CR cells are unchanged in midline tissue of Foxj1Cre; β-Catenin GOF brains at E14.5. (G) TRP73 and REELIN staining co-localizes with Ai9+ cells in the hippocampal fissure in both control and Foxj1Cre ; β-Catenin GOF brains at E16.5 and E18.5. Scatterplots in (C) and (F) display Mean ± SEM. Statistical test (C): Shapiro-Wilk normality test, followed by Welch’s two sample t-test, (F) Multiple Mann-Whitney Tests; p < 0.0001; *p < 0.05; **p < 0.01; ***p < 0.001; ns if p value > 0.05. For (C), N=3 (biologically independent replicates), (E), N=5 (biologically independent replicates), (F), N=6 (biologically independent replicates), (G), N=3 (biologically independent replicates). Scale bars: 100 μm (all images in C, E and G).

Article Snippet: Primary antibodies used: Lef1(rabbit, 1:200, CST catalogue #C12A5), β-CATENIN (Mouse, 1:200, BDbiosciences catalogue #610153), β-CATENIN (Rabbit, 1:50, CST catalogue # 8814), RFP (rabbit, 1:200, Abcam catalogue #ab62341), RFP (Mouse, 1:200, Invitrogen catalogue #MA5-15257), β-III TUBULIN (mouse, 1:100, Promega catalogue #G7128), TRP73 (Rabbit, 1:200, CST catalogue #14620S), REELIN (Mouse, 1:200, Millipore catalogue #MAb5364), NEUN (Rabbit, 1:200, invitrogen catalogue #702022).

Techniques: Control, Derivative Assay, Expressing, Staining, Activity Assay, MANN-WHITNEY, IF-P