rabbit anti p73  (Bethyl)

Journal: bioRxiv

Article Title: Differential contribution of P73 + Cajal-Retzius cells and Reelin to cortical morphogenesis

doi: 10.1101/2024.10.15.618167

Figure Lengend Snippet: (A) Gene expression in E12 CRs subtypes and other glutamatergic neurons from the dorsal and lateral cortex (extracted from https://apps.institutimagine.org/mouse_pallium/ ). (B) Expression of Nhlh2 per cell type and stage in scRNAseq data from the somatosensory cortex. Grey squares indicate no cells were sampled. Note that Nhlh2 expression is restricted to CRs except at early stages where it is also detected in intermediate progenitors and immature neurons. (C, D) In situ hybridization for Nhlh2 on coronal sections of the cerebral cortex from E14 (C) and E18 (D) control and Gmnc -/- embryos. The presence/absence of Nhlh2 + cells in the MZ is indicated by filled/empty arrowheads, respectively. (E) High magnification of the dorsal or lateral cortex MZ after in situ hybridization for Tbr1 and Calb2 at E18, and Lhx5 at P1. (F) In situ hybridization for Nhlh2 and Trp73 on coronal sections of the E18 hippocampus. (G) In situ hybridization for Trp73 on coronal sections of the E14 dorsomedial cortex. (H) Immunostaining for P73 on coronal sections of the E18 hippocampus and dorsomedial cortex. (I) Quantification of the density of P73 + cells in the hippocampal and neocortical MZ of E18 control and Gmnc -/- embryos. Each dot corresponds to one measurement, 3 animals (color-coded) and 3 rostro-caudal levels (shape) were considered. (J) Immunostaining for P73, and Tomato in the hippocampus at P0 following genetic tracing of hem derivatives in a Gmnc -/- background, showing residual hem-derived CRs in mutants. (K) Immunostaining for Tomato and DAPI in the dorsal cortex at P0 following genetic tracing of hem derivatives in either control or Gmnc -/- background, showing the complete absence of Tomato + cells in the mutant neocortical MZ. (L) Schematic representation of the temporal dynamics of CRs depletion in Gmnc -/- mutants. Scale bars: 200µm in C, F, G, H, 500µm in D, 50µm in high magnification panels in C, D, E and in J, K.

Article Snippet: The following primary antibodies were used: goat anti-Brn2 (POU3F2, Abcam ab101726 1:1000), rat anti-CTIP2 (BCL11B, Abcam ab18465 1:600), rabbit anti-FOXG1 (Abcam ab18259 1:2000), rabbit anti-Laminin (Sigma-Aldrich L9393 1:600), goat anti-Neuropilin-1 (R&D Systems AF566 1:800), rabbit anti-p73 (Cell signaling 14620 1:250), goat anti-Nurr1 (NR4A2, R&D Systems AF2156 1:200), goat anti-Prox1 (R&D Systems AF2727 1:1000), goat anti-Reelin (R&D Systems AF3820 1:2000), rabbit anti-TBR1 (Abcam ab31940 1:1000).

Techniques: Expressing, In Situ Hybridization, Control, Immunostaining, Derivative Assay, Mutagenesis

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used: sheep polyclonal anti-GPR64 (AF7977, R & D Systems); rabbit polyclonal anti-GPR64 C terminus (GTX70517, GeneTex); goat polyclonal anti-SMPD1 (AF5348, R & D Systems); mouse monoclonal anti-tubulin (DM1A, Thermo Fisher Scientific); mouse monoclonal anti-FLAG (F1804, Sigma-Aldrich); rabbit polyclonal anti-FLI1 (ab15289, Abcam), rabbit monoclonal anti-HA (3724, Cell Signaling Technology); rabbit monoclonal anti-Phospho-CREB (Ser133) (9198, Cell Signaling Technology); rabbit monoclonal anti-CREB (9197, Cell Signaling Technology); rabbit monoclonal anti-RIF1 (95558, Cell Signaling Technology); rabbit polyclonal anti-SPOP (16750-1-AP, Proteintech); mouse monoclonal anti-p21 (2946, Cell Signaling Technology); mouse monoclonal anti-p27 (sc-528, Santa Cruz Biotechnology); rabbit polyclonal antibody anti-p16 (sc-468, Santa Cruz Biotechnology); rabbit monoclonal anti-p73 (14620, Cell Signaling Technology); rabbit monoclonal anti-β3-Tubulin (5568, Cell Signaling Technology); rabbit monoclonal anti-Neurofilament-L (2837, Cell Signaling Technology); rabbit monoclonal anti-caspase-3 (9665, Cell Signaling Technology); mouse monoclonal anti-PARP1 (9542,Cell Signaling Technology); anti-rabbit IgG, HRP-linked antibody (7074, Cell Signaling Technology); anti-mouse IgG, HRP-linked antibody (7076, Cell Signaling Technology); rabbit anti-goat IgG HRP-linked antibody (HAF017, R & D Systems); and donkey anti-Sheep IgG HRP-linked antibody (HAF016, R & D Systems).

Techniques: Control, Virus, Recombinant, Transfection, SYBR Green Assay, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Plasmid Preparation

Journal: Frontiers in Neuroscience

Article Title: Differentiation stage-specific expression of transcriptional regulators for epithelial mesenchymal transition in dentate granule progenitors

doi: 10.3389/fnins.2024.1425849

Figure Lengend Snippet: Scrt2 is expressed in Tbr2+ GNPs but not in p73+/Reelin+ Cajal–Retzius neurons. Scrt2 is expressed in Tbr2+ GNPs but not in p73+ cells at E14, E16, and P6 (arrows in A7, B7, C7, respectively). P73 is expressed in reelin+ Cajal–Retzius neurons but not in gfap -GFP+ cells at E18 (arrows in D1–D8 ). The box in panels (A1,B1,C1,D1) indicates the region shown in panels (A2–A8,B2–B8,C2–C8,D2–D8) . Scale bars; 200 μm in (A1,B1,C1,D1) ; 50 μm in (A2–A8,B2–B8,C2–C8,D2–D8) .

Article Snippet: The following primary antibodies were used: anti-GFP (abcam, ab13970, 1:5,000); anti-Lhx1/5 mouse IgG (DSHB, 4F2, 1:50); anti-Neuro D goat IgG (N-19) (Santa Cruz, sc-1084, RRID:AB_630922, 1:1,000); anti-Nkx6-2 (Millipore, ABN1455); anti-p73 goat IgG (Santa Cruz, sc-9651, 1:250); anti-Prox1 goat IgG (R&D, AF2727, 1:1,000); anti-Tbr2/EOMES rat IgG conjugated to Alexa 660 (eBioscience, 50-4375-82, 1:1,000); mouse anti-reelin (Millipore, MAB5364, clone G10, 1:1,000); anti-Scratch2 (Scrt2) rabbit polyclonal antibody (gift of Y Gotoh; 1:1,000); anti-Sox2 goat IgG (R&D, AF2018, 1:1,000); anti-Zeb1 rabbit IgG (Thermo Fischer Scientific/Sigma HPA027524, 1:1,000); anti-Zeb1 mouse IgG (eBioscience, 14-9741-80, 1:250).

Techniques:

Journal: bioRxiv

Article Title: Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling

doi: 10.1101/2024.05.31.596474

Figure Lengend Snippet: ( A ) Representative immunoblot image and quantification of YAP protein levels phosphorylated at the tyrosine residue 357 (pTyr357-YAP) in Wt and CLN3 KO cells. GAPDH and Ponceau were used as loading controls. ( B ) Confocal fluorescence images of Wt and CLN3 KO cells immunostained for pTyr357-YAP protein. Nucleus are outlined by the yellow dashed line using Hoechst staining. Scale bar 10µm. On the right, quantification of the mean intensity of YAP at the nucleus of at least 500 cells per each condition. The yellow dots represent the mean of each independent experiment. ( C ) Immunohistochemistry analysis of pTyr357-YAP staining of control (Wt) and Cln3 Δex7/8 animals. On the right, quantification of the number of positive nucleus to pTyr357-YAP per the same area of hippocampus (first) and thalamus (second). ( D ) Representative immunoblot image and quantification of pTyr357-YAP in Wt and Cln3 Δex7/8 animals. GAPDH and Ponceau were used as loading controls. ( E ) Confocal fluorescence images of Wt and CLN3 Kd cells immunostained for p73 protein. Nuclei are outlined by the yellow dashed lines using Hoechst staining. Scale bar 10µm. On the right, quantification of the mean intensity of p73 at the nucleus of at least 500 cells per each condition. ( F ) Correlation between the intensity levels of nuclear YAP (x axis) and p73 (y axis) proteins. At least 150 cells were analysed. ( G ) Confocal fluorescence images and quantification of HEK293T (upper panels and graph) and ARPE19 (bottom panels and graph) parental cell lines treated with scramble or CLN3-siRNA and immunostained for p73 protein. Nuclei are outlined by the yellow dashed line using Hoechst staining. Scale bar 10µm. All the results are mean±SEM of at least three independent experiments. ( H ) Confocal fluorescence images of CLN3 KO cells transfected with a control or two siRNA against YAP protein and immunostained for p73 protein. Nuclei are outlined by the yellow dashed lines using Hoechst staining. Scale bar 10µm. On the right, quantification of the mean intensity of p73 at the nucleus of at least 450 cells per each condition. ( I ) Representative immunoblot image and quantification of p73 co-immunoprecipitated with YAP protein in Wt and CLN3 Kd cells. The results are mean±SEM of four independent experiments. In the violin plots, yellow dots represent the mean of each individual experiment. Statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparisons test ( G and H ), and Student’s t -tests when comparing two experimental groups. * p<0.05; ** p<0.01; ***p<0.001. * p<0.05; *** p<0.001; **** p<0.0001.

Article Snippet: The following antibodies were used: anti-YAP (Cell Signalling, 4912S; IB 1:1000); anti-pTyr357-YAP (Sigma, Y4645; IB 1:1000, IF 1:100); anti-p73 (Cell Signaling Technology, 14620S; IB 1:1000, IF 1:100); anti-cAbl (Cell Signalling 2662P; IB 1:1000, IF 1:100); anti-H2Ax (Cell Signalling, 9718S; IB 1:1000, IF 1:100); anti-p53 (Proteintech, 10442-1-AP; IB 1:1000, IF 1:100); anti-p-p53 (Cell Signalling, 9286P; 1:1000, IF 1:100); anti-mouse monoclonal anti-Tubulin (Sigma-Aldrich, T6199; IB 1:1000); anti-rabbit monoclonal anti-GAPDH (Cell signalling, 5174T; IB 1:1000); mouse polyclonal anti-hnRNPA2B1 [B-7] (Santa Cruz, sc-374053; IB:1000); anti-cleaved-PARP (Cell Signalling, 5625P, IB 1:1000); anti-ATM (Cell Signalling, 2873; IB 1:1000); anti-p-ATM (Ser1981) (D6H2) (Cell Signalling, 5883; IB 1:1000); anti-lamin A/C (Santa Cruz, 376248; IF 1:100); HRP-conjugated goat anti-mouse IgG (Cell Signalling, 7076S; IB 1:5000); HRP-conjugated goat anti-rabbit IgG (Cell Signalling, 7074P2; IB 1:5000); Alexa647-conjugated goat anti-rabbit IgG (Invitrogen, A21245; IF 1:500); Alexa568-conjugated goat anti-mouse IgG (Invitrogen, A11031; IF 1:500); mouse monoclonal anti-LAMP1 [H4A3] (development studies hybridoma bank, AB_2296838; IB 1:1000, IF 1:100); anti-CLN3 (kindly given by Alessia Calcagni’ ( ); anti-LC3 (ab48394; IB 1:1000, IF 1:100); anti-PLA2G15 (Proteintech, 10863-2-AP; IB 1:1000).

Techniques: Western Blot, Residue, Fluorescence, Staining, Immunohistochemistry, Control, Transfection, Immunoprecipitation

Journal: Pharmaceutics

Article Title: The Proteasome Inhibitor CEP-18770 Induces Cell Death in Medulloblastoma

doi: 10.3390/pharmaceutics16050672

Figure Lengend Snippet: Synergistic effect between cisplatin pre-treatment and CEP-18770 in MBs cells. ( A ) DAOY and ( B ) UW228-2 cells were treated with different concentrations of cisplatin first, and after 10 h CEP-18770 was added. We tested two concentrations of CEP-18770, 7.5 pM and 15 pM. The cells were incubated for a further 30 h and cell viability was determined by Cell TiterGlo assay. ( C ) Western blot showing p73 levels in DAOY cells after cisplatin pre-treatment and 7.5 pM CEP-18770 was added. Two concentrations of cisplatin were tested: 80 and 160 nM. Tubulin was used as a loading control. * p < 0.05, ** p < 0.001; *** p < 0.0001.

Article Snippet: We used rabbit anti-p73 (1/1000) (polyclonal antibody, A300-126A, Bethyl Laboratories Inc., Montgomery, TX, USA) and rabbit anti-Ubiquitin P4D1 (1/1000) (monoclonal antibody, Cell Signaling Technologies, Danvers, MA, USA).

Techniques: Incubation, Western Blot, Control