monoclonal rat anti ptch1 antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Inhibition of PTCH1 drug efflux activity enhances chemotherapy efficacy against triple negative breast cancer

doi: 10.1101/2025.09.03.673759

Figure Lengend Snippet: A . Relative PTCH1 RNA expression in TNBC (red), Her2 (blue), luminal B (LB, green), and luminal A (LA, orange) in the TCGA cohort is illustrated by box plots (log 2 transformed). Outliers are shown within each population (open circles). Student’s t test was used to compare RNA levels between two groups. The p values are indicated (*P < 0.05; **P < 0.01; ***P < 0.001; ns P > 0.05). B . Normalized PTCH1 mRNA expression according to TNBC (IHC) status from all DNA microarray data from bc-GenExMiner v4.5 illustrated by box plots (log 2 transformed) (Jezequel et al 2021). C . Boxplots showing PTCH1 mRNA expression (normalized counts from RNA seq data) in circulating tumor cells (CTC) isolated from metastasis breast cancer patient peripheral blood (Wurth R et al 2025). D . High PTCH1 mRNA expression is associated with a poorer prognosis in breast cancer. Distant Metastasis Free Survival (DMFS) and disease-free survival (DFS) data based on PTCH1 mRNA expression were obtained using the exhaustive prognostic analysis on all status breast cancers on the bc-GenExMiner v4.5 web portal, and illustrated by Kaplan–Meier (KM) curves for all ER and PR breast cancers with positive nodules. The obtained Hazard Ratio (HR) with 95% confidence interval and log-rank p-values are shown.

Article Snippet: After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1 % Tween-20, and 5 % non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with the monoclonal rat anti-PTCH1 antibody from R&D system biotechne clone 413220 (1/1000), the monoclonal mouse anti-P-gp antibody from abcam (ab3366, 1/1000), the rabbit anti-ABCG2 antibody from GeneTex (GTX50793, 1/500) or the rabbit anti-GAPDH antibody from Elabscience (1/20000).

Techniques: RNA Expression, Transformation Assay, Expressing, Microarray, RNA Sequencing, Isolation

Journal: bioRxiv

Article Title: Inhibition of PTCH1 drug efflux activity enhances chemotherapy efficacy against triple negative breast cancer

doi: 10.1101/2025.09.03.673759

Figure Lengend Snippet: Distant Metastasis Free Survival (DMFS), disease-free survival (DFS) and overall survival (OS) data based on PTCH1 mRNA expression were obtained from the intrinsic molecular subtypes’ prognostic analysis on ER+/HER2-high proliferative breast cancers performed on bc-GenExMiner v4.5 web portal and illustrated by Kaplan–Meier curves. The obtained Hazard Ratio (HR) with 95% confidence interval and log-rank P-values are shown.

Article Snippet: After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1 % Tween-20, and 5 % non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with the monoclonal rat anti-PTCH1 antibody from R&D system biotechne clone 413220 (1/1000), the monoclonal mouse anti-P-gp antibody from abcam (ab3366, 1/1000), the rabbit anti-ABCG2 antibody from GeneTex (GTX50793, 1/500) or the rabbit anti-GAPDH antibody from Elabscience (1/20000).

Techniques: Expressing

Journal: bioRxiv

Article Title: Inhibition of PTCH1 drug efflux activity enhances chemotherapy efficacy against triple negative breast cancer

doi: 10.1101/2025.09.03.673759

Figure Lengend Snippet: A . PTCH1 mRNA expression (log 2 transformed) in various luminal and HER2 breast cancer cell lines (dark green) and in TNBC cell lines (other colors). TNBC cell lines are depicted according to the “Lehmann TNBC subtype” nomenclature : basal-like 1 (yellow), basal-like 2 (pale green), immunomodulatory (brown), luminal androgen receptor (dark pink), mesenchymal (pale pink) and mesenchymal stem-like (pink). B . PTCH1 protein expression in three TNBC cell lines. Western-blot was performed on 50 µg extracts from TNBC cell lines (MDA-MB-231, HCC-38 and MDA-MB-468) with antibodies directed against PTCH1. PTCH1 and GAPDH signals were quantified using ImageJ software. Data presented are the mean ± SEM of at least 3 independent experiments. Significance is calculated using Oneway ANOVA Turkey’s multiple comparisons test and attained at P < 0.05 (*).

Article Snippet: After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1 % Tween-20, and 5 % non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with the monoclonal rat anti-PTCH1 antibody from R&D system biotechne clone 413220 (1/1000), the monoclonal mouse anti-P-gp antibody from abcam (ab3366, 1/1000), the rabbit anti-ABCG2 antibody from GeneTex (GTX50793, 1/500) or the rabbit anti-GAPDH antibody from Elabscience (1/20000).

Techniques: Expressing, Transformation Assay, Western Blot, Software

Journal: bioRxiv

Article Title: Inhibition of PTCH1 drug efflux activity enhances chemotherapy efficacy against triple negative breast cancer

doi: 10.1101/2025.09.03.673759

Figure Lengend Snippet:

Article Snippet: After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1 % Tween-20, and 5 % non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with the monoclonal rat anti-PTCH1 antibody from R&D system biotechne clone 413220 (1/1000), the monoclonal mouse anti-P-gp antibody from abcam (ab3366, 1/1000), the rabbit anti-ABCG2 antibody from GeneTex (GTX50793, 1/500) or the rabbit anti-GAPDH antibody from Elabscience (1/20000).

Techniques: Concentration Assay

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 2. Expression of Shh pathway genes in neurospheres from euploid and Ts65Dn mice. (A–C) Quantification by RT–qPCR of Gli1, Gli2 and Gli3 (A); MycN, Bmi1 and FoxM1 (B); Shh, Ptch1 and Smo (C) expression in neuro- spheres from Ts65Dn (n ¼ 6) and euploid (n ¼ 6) mice, given as percentage of the control condition. (D) Western blot analysis of Ptch1 expression in neu- rospheres from Ts65Dn (n ¼ 4) and euploid (n ¼ 4) mice. Protein expression was normalized to b-actin content. Values represent mean+SE. ∗P , 0.05, ∗∗P , 0.01, ∗∗∗P , 0.001 (two-tailed t-test). (E) Representative images of neurospheres from an euploid and a Ts65Dn mouse processed for Ptch1 fluor- escence immunocytochemistry. Cell nuclei were stained by Hoechst dye (blue). Scale bar: 20 mm.

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Two Tailed Test, Immunocytochemistry, Staining

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 3. Response to Shh in neurospheres from euploid and Ts65Dn mice. (A) LI, defined as percentage of BrdU-positive cells over total cell number, was determined for neurospheres from euploid (n ¼ 3) and Ts65Dn (n ¼ 3) mice. Neurospheres were treated with Shh (3 mg/ml), cyclopamine (Cyc; 10 mg/ml), SAG (250 nM), Shh plus cyclopamine or SAG plus cyclopamine for 72 h starting from DIV1. (B) Quantification by RT–qPCR of Gli1 expression in neurospheres treated with Shh (3 mg/ml) or SAG (250 nM) for 24 h starting at DIV4 (three Ts65Dn and three euploid mice). Data are given as percentage of the euploid untreated condition. The scheme on the right shows that Shh drives the Shh pathway by removing the inhibition exerted by Ptch1 on Smo, and the negative and positive effect exerted by Cyc and SAG, respectively, on Smo. Values represent mean+SE. The aster- isk indicates a significant difference in euploid and trisomic neurospheres versus euploid untreated neurospheres. The # indicates a significant difference of trisomic treated neurospheres versus trisomic untreated neurospheres. ∗P , 0.05; ∗∗P , 0.01; ∗∗∗P , 0.001 (Bonferroni’s test after ANOVA).

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Quantitative RT-PCR, Expressing, Inhibition

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 4. Effect of Ptch1 silencing on cell proliferation in neurospheres from euploid and Ts65Dn mice. (A) Quantification of Ptch1 immunofluorescence in neurospheres from Ts65Dn and euploid mice after 24 h exposure to a Ptch1 antisense oligonucleotide (10 mM) starting at DIV3 (left panel). Representative example of a western blot showing the Ptch1 band in cell extracts from triso- mic and euploid neurospheres after 24 h exposure to a Ptch1 antisense oligo- nucleotide (right panel). The partial silencing of Ptch1 by antisense oligonucleotides is shown in the scheme on the right. (B) LI, defined as per- centage of BrdU-positive cells over total cell number, was determined for euploid and trisomic neurospheres treated with Ptch1 antisense oligonucleo- tides (10 mM) for 72 h after DIV1. (C) LI determined for euploid and trisomic neurospheres treated with either Shh alone (3 mg/ml; L and 6 mg/ml; H) or Shh (3 mg/ml; L) plus Ptch1 antisense oligonucleotides (10 mM) for 72 h after DIV1. Data in (A, B, C), given as percentage of the euploid untreated con- dition, are expressed as mean+SE (four euploid and four Ts65Dn mice). The asterisks indicate a significant difference of euploid and trisomic neuro- spheres versus euploid untreated neurospheres. ∗∗P , 0.01, ∗∗∗P , 0.001 (Bonferroni’s test after ANOVA).

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Western Blot

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 5. Methylation and acetylation status of the Ptch1 promoter in neuro- spheres from euploid and Ts65Dn mice. (A) Quantification by RT–qPCR of Ptch1 expression in neurospheres from Ts65Dn (n ¼ 4) and euploid (n ¼ 4) mice treated with either 5-Aza-2′-deoxycytidine (AZA, 5 mM) or TSA (150 nM) for 72 h after DIV1. Data, given as percentage of the euploid untreated condition, are expressed as mean+SE. The asterisks indicate a sig- nificant difference between euploid and trisomic neurospheres versus euploid untreated neurospheres. ∗∗P , 0.01, ∗∗∗P , 0.001 (Bonferroni’s test after ANOVA). (B) The mCIP assay (upper panels) was performed with an anti- methylcytosine antibody and primers targeting amplicons a–e in neurospheres at DIV4 from trisomic (n ¼ 4) and euploid (n ¼ 4) mice. Fold enrichment of the Ptch1 gene promoter by anti-methylcytosine antibody was calculated by dividing the PCR product from amplicons b, c or e by the PCR product from amplicons a and d that represent non-CpG control regions. The ChIP assay (middle panels) was performed using an anti-Acetyl-Histone H3 and the same primers used for the mCIP. Fold enrichment of a given DNA region immunoprecipitated with the anti-Acetyl-Histone H3 antibody was cal- culated as the ratio between the enrichment obtained with the specific antibody compared with pre-immune serum. Values represent mean+SE. ∗P , 0.05, ∗∗P , 0.01, ∗∗∗P , 0.001 (two-tailed t-test). The lower panel shows a sche- matic representation of the mouse Ptch1 gene promoter. The diagram includes: transcription start site (black arrow); target regions of PCR primers for quan- titative ChIP assay (amplicons a–e: gray boxes); CpG-rich region and CpG islands (black boxes).

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Methylation, Quantitative RT-PCR, Expressing, Control, Immunoprecipitation, Two Tailed Test

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 6. AICD expression and binding to Ptch1 promoter in neurospheres from euploid and Ts65Dn mice. (A) Quantification by RT–qPCR of APP expression in trisomic and euploid neurospheres at DIV4. Values represent mean+SE. ∗∗P , 0.01 (two-tailed t-test). (B) Representative examples of western blots showing the AICD 6 kDa band in nuclear fractions from trisomic and euploid neurospheres. (C) The ChIP assay using an anti-C-terminal APP antibody which recognizes AICD and the same primers used for the mCIP (amplicons a–e are the same as Fig. 5B) in neurospheres from trisomic (n ¼ 4) and euploid (n ¼ 4) mice in the presence of absence of the g-secretase inhibitor DAPT (10 mM; 48 h). Fold enrichment of a given DNA region of the mouse Ptch1 gene promoter was calculated as the ratio between the enrich- ment obtained with the specific antibody compared with a non-specific IgG (control). (D) Quantification of Ptch1 immunofluorescence in neurospheres from Ts65Dn and euploid mice after 48 h exposure to the g-secretase inhibitor DAPT (10 mM) or Compound E (Comp. E; 1 nM), starting at DIV1. The aster- isk in (C and D) indicates a significant difference of euploid and trisomic neu- rospheres versus euploid untreated neurospheres. The # indicates a significant difference of trisomic treated neurospheres versus trisomic untreated neuro- spheres. ∗P , 0.05; ∗∗P , 0.01; ∗∗∗P , 0.001 (Bonferroni’s test after ANOVA).

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Two Tailed Test, Western Blot, Control

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 7. Ptch1 expression in Ts65Dn mice. (A) Examples of Ptch1 fluor- escence immunohistochemistry at the level of the rostral part of the lateral ventricle of a P2 euploid and a Ts65Dn mouse. Images were taken in the regions labeled with the corresponding numbers in the section shown in the middle (1, neocortex; 2, striatum; 3, roof of the lateral ventricle; 4, lateral wall of the lateral ventricle). Calibrations: low magnification image ¼ 200 mm; higher magnification images ¼ 40 mm. (B) Quantification of Ptch1 expression in euploid and Ts65Dn mice. (C) Examples of Ptch1 fluorescence immunohistochemistry at the level of the hippocampal DG (left panels). Scale bar: 60 mm. (D) Quantification of Ptch1 expression in the granule cell layer (GR) and hilus (SGZ plus hilus proper) of euploid and Ts65Dn mice. Data (in B and D), given as percentage of the euploid condition, are expressed as mean+SE (three euploid and three Ts65Dn mice). ∗P , 0.05, ∗∗P , 0.01, ∗∗∗P , 0.001 (two-tailed t-test). GR, granule cell layer; H, hilus; I, layer one; II layer two; LW, lateral wall of the lateral ventricle; ML, medial wall of the lateral ventricle; NC, neocortex; ROOF, roof of the lateral ventricle; ST, striatum.

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Expressing, Immunohistochemistry, Labeling, Two Tailed Test

Journal: Human molecular genetics

Article Title: APP-dependent up-regulation of Ptch1 underlies proliferation impairment of neural precursors in Down syndrome.

doi: 10.1093/hmg/ddr033

Figure Lengend Snippet: Figure 8. PTCH1 expression in control and DS human fetuses. (A) Examples of PTCH1 fluorescence immunohistochemistry at the level of the hippocampal region of a control (GW 19) and a DS (GW 19) fetuses. Images were taken in the regions labeled with the corresponding numbers in the Nissl-stained section shown in the middle. Calibrations: low magnification image ¼ 1000 mm; higher magnification images ¼ 50 mm. DG, dentate gyrus; FI, fimbria; GM, germinal matrix; HIPP, hippocampus; PHG, parahippocampal gyrus; TH, temporal horn of the lateral ventricle. (B) Quantification of PTCH1 expression in control and DS fetuses. Data, given as percentage of the control condition, are expressed as mean+SE (three control and three DS fetuses). ∗∗P , 0.01 (two-tailed t-test).

Article Snippet: For Ptch1 immunocytochemistry, we used an anti-Ptch1 rat monoclonal antibody (1:50; R&D systems) and, as a secondary antibody, a Cy3-conjugated anti-rat antibody (1:200; Jackson Laboratories).

Techniques: Expressing, Control, Immunohistochemistry, Labeling, Staining, Two Tailed Test