Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: The list of antibodies used for Western blotting.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: The sequences of primers used for real-time PCR.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: (A) Decreasing mRNA expression levels of RASAL1 upon the time of high Pi treatment after 24, 48 and 72 hours. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, *P<0.05, **P<0.01, ***P<0.001). (B) Western blot confirming the decreased protein expression of RASAL1 in Pi treated cells. (C) MeDIP result showing the methylated promoter of RASAL1 along the treatment time, correlated with the reduced expression of RASAL1 in both mRNA and protein level. (D) Ras activity was measured by ELISA assay, untreated cells served as controls. Pi- treated cells showed the hyper-activation of total Ras.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Expressing, Western Blot, Methylated DNA Immunoprecipitation, Methylation, Activity Assay, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: Cells were treated with Pi 3mM for 72 hours. (A) MeDIP result showing the demethylated promoter of RASAL1 by using PFA, correlated with restored mRNA expression of RASAL1. (B) qPCR analysis showed the restored RASAL1 mRNA expression levels by using phosphate transporter inhibitor (PFA). Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, **P<0.01). (C) ELISA assay indicating the normalization of total Ras with combination of PFA or RAS inhibitor farnesylthiosalicylic acid (FTS). (D) bright-field images showing the morphology change of HCAEC cells cultured under normal control condition, high Pi conditions, and Pi combined with FTS or PFA. Scale bars 25 μm. (E) Western blot analysis showing the expression of endothelial cell marker CD31 and fibroblast cell marker S100A4 in HCAEC cells exposed to normal control condition, high Pi conditions, and Pi combined with FTS or PFA (F) qRT-PCR data showing the mRNA expression levels of EndMT transcriptional factors (SNAIL, SLUG, and TWIST) and FSP1 in HCAEC cells under four conditions indicated above. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, ***P<0.001).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Methylated DNA Immunoprecipitation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Western Blot, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: (A) Simplified schematic showing the human RASAL1 promoter along with exons (black boxes), translational start site (black arrow), locations of RASAL1 chip primer for amplicon1 and amplicon2, and amplicon2 primers serve as negative control chip primer. (B-C) The binding properties of HDAC2 and DNMT1 to the RASAL1 promoter region were analyzed by chromatin immunoprecipitation (ChIP) assay and detected by qRT-PCR in Pi treated (B) or in control cells (C). IgG purified from the same species serve as negative control for ChIP (expression are presented as means ± s.d., n = 3 independent experiments, **P<0.01, ***P<0.001, n.s. no significance).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Negative Control, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, Purification, Expressing

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: In the physiological conditions, the CpG islands located in RASAL1 promoter are unmethylated (top panel) as indicated by open circles, and RASAL1 is transcriptional active. Under pathological conditions, initially when endothelial cells are exposed to stimulus, such as TGFβ1 or high concentration of Pi resulting in RASAL1 silencing through condensed chromatin structure at RASAL1 promoter mediated by HDAC2. While the RASAL1 promoter remains unmethylated (middle panel) and RASAL1 is transiently silenced. When the cells are continuously exposed to the stimulus, the CpG islands located in RASAL1 promoter are methylated (indicated by filled circles) by DNMT1 recruited through the interaction with HDAC2. Therefore, RASAL1 is permanently silenced due to promoter hypermethylation (lower panel).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Concentration Assay, Methylation

Journal: The Journal of cell biology

Article Title: Centrobin controls primary ciliogenesis in vertebrates.

doi: 10.1083/jcb.201706095

Figure Lengend Snippet: Figure 2. CNTROB null cells are defective in primary ciliogenesis. (A) Localization of centrobin (green) in asynchronous (Asynch.) or 48-h serum-starved cells. Detyr. Tub, detyrosinated tubulin. Bars: 5 µm; (inset) 1 µm. (B) IF microscopy of the cilium markers ARL13B (green) and acetylated tubulin (red) in cells after 48-h serum starvation. Bars: 5 µm; (inset) 2 µm. (C) Quantitation of the ciliation frequency after 48-h serum starvation showing mean + SEM of three independent experiments in which at least 100 cells were quantitated by acetylated tubulin staining. **, P < 0.01; ***, P < 0.001, in comparison to indicated samples by unpaired t test. (D) siRNA knockdown of CNTROB was used to ablate centrobin, with a GAPDH siRNA used as negative control (siGAPDH). Bar chart shows quantitation of the ciliation frequency after 24 h serum starvation. (E) Quantitation of the ciliation frequency after 48-h serum starvation of cells transiently transfected with GFP-tagged centrobin constructs. Bar charts show mean + SEM of three independent experiments in which at least 100 cells were quantitated by ARL13B staining. *, P < 0.05; **, P < 0.01, in comparison to controls by unpaired t test. (F) IF microscopy of U2OS cells transiently transfected with GFP-tagged human or Drosophila centrobin (green) with pericentrin (red) as a marker for centrosomes. Bars: 5 µm; (inset) 1 µm. (G) Transmission electron microscopy analysis of WT and centrobin null (knockout [KO]) cells after 48-h serum starvation. Bars, 500 nm.

Article Snippet: The primary antibodies used in this study were as follows: centrobin (6D4F4; 1:1,000), CP110 (1:2,000; 12780-1-AP; ProteinTech), α-tubulin (1:10,000; B512; Sigma-Aldrich), GFP (1:15,000; 66002- 1-Ig; Proteintech), and GAP DH (1:10,000; 2118; Cell Signaling).

Techniques: Microscopy, Quantitation Assay, Staining, Comparison, Knockdown, Negative Control, Transfection, Construct, Marker, Transmission Assay, Electron Microscopy, Knock-Out

Journal: The Journal of cell biology

Article Title: Centrobin controls primary ciliogenesis in vertebrates.

doi: 10.1083/jcb.201706095

Figure Lengend Snippet: Figure 3. The N-terminal CPAP-binding region of centrobin is dispensable for ciliogenesis. (A) Schematic of deletion analysis performed on centrobin, indicating regions required for CPAP, CEP152, or tubulin interactions or localization to the centrosome. (B) Immunoblot showing expression of each of the GFP-tagged centrobin fragments at 24 h after transfection into HCT116 cells. The control lane is from untransfected cells. The lane with full-length protein is overloaded relative to the others. Nsp, nonspecific band. (C) Fluorescence micrographs of CNTROB null cells 24 h after transfection with GFP-tagged expression constructs that were tested in B. In the merged images, GFP localization is shown in green, ARL13B in red, and centrin2 in magenta. The same order is used for all single channels in the blowups. Bars: 5 µm; (inset) 2 µm. (D) Quantitation of the frequency of primary cilia in CNTROB null cells transfected with the indicated constructs. Cells were serum starved for 24 h, starting 24 h after transfection. Scoring was based on ARL13B staining and bar chart shows mean + SEM from 100 transfected cells in each of three experiments. **, P < 0.01; ***, P < 0.001, compared with full-length rescue using one-way ANOVA and Tukey’s multiple comparison test. (E) IF microscopy of CP110 and CEP97 localization in WT and centrobin-null hTERT-RPE1 cells. Bars: 5 µm; (inset) 2 µm. (F) Coimmunoprecipitation (Co-IP) of endogenous CNTROB and CP110 from hTERT-RPE1 cell extract using anticentrobin monoclonal antibody 6D4F4 for the pulldown. CNTROB-null cells were used as the negative control. (G) Coimmunoprecipitation experiment using poly- clonal anti-CP110 for pulldown 24 h after transfection of HCT116 WT cells with the indicated GFP constructs. IgG incubated with cell extracts was used as negative control. Size markers are in kilodaltons.

Article Snippet: The primary antibodies used in this study were as follows: centrobin (6D4F4; 1:1,000), CP110 (1:2,000; 12780-1-AP; ProteinTech), α-tubulin (1:10,000; B512; Sigma-Aldrich), GFP (1:15,000; 66002- 1-Ig; Proteintech), and GAP DH (1:10,000; 2118; Cell Signaling).

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Control, Fluorescence, Construct, Quantitation Assay, Staining, Comparison, Microscopy, Co-Immunoprecipitation Assay, Negative Control, Incubation

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: A UMAP plots of epithelial cells from primary TNBC tumours showing correlation between CytoTRACE score (1—least differentiation, 0 – most differentiation) and RASAL2 expression. B Violin plots of RASAL2 expression and residual tumour signature expression in the four clusters of epithelial cells identified in the TNBC tumours in ( A ). Cluster 1 expressions are significantly higher compared to all other clusters. Data are represented as mean ± SEM. P value by one-way ANOVA. C Heatmap of RASAL2 expression in pre- and post-treatment breast cancer patients . Fold change (FC) was determined relative to pre-treatment expression level. P value by paired T -test. D Dot plots of RASAL2 expression in pre- versus post-treatment TNBC/ BRCA -mutant breast cancer patients . Probes that recognise RASAL2 variant 2 (ILMN_1813701 and ILMN_1673455) show significant increase following treatment. Data are represented as mean ± SEM. P value by two-tailed T -test. E Immunohistochemistry of fixed post-treatment TNBC patient breast specimens. RASAL2 (brown stain) was enriched in the tumour compartment versus adjacent normal epithelia. Data are represented as mean ± SEM. P value by two-tailed T -test. Scale bar, 20 µm. F Immunoblotting of fresh post-treatment TNBC patient specimens. RASAL2 was enriched in the tumour (T) versus adjacent normal (N) tissues in TNBC patients. LE long exposure.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Mutagenesis, Variant Assay, Two Tailed Test, Immunohistochemistry, Staining, Western Blot

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: A Correlation between RASAL2 expression and sensitivity to indicated chemotherapy. The area under percent-viability curves (AUC) was computed as a metric of drug sensitivity, as derived from the Cancer Therapeutics Response Portal. Pearson r and P value are reported. B Cell viability assay. Vector control and RASAL2-overexpressing MDA-MB-468 cells were treated as indicated. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T -test. C Spheroid assay. Viability of vector control and RASAL2-overexpressing TNBC spheroids was measured following treatment with vehicle DMSO, doxorubicin (DOXO) or gemcitabine (GEM). Representative images of HCC1806 spheroids are shown. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T -test. Scale bar, 250 µm. Cell viability assay. Vector control and RASAL2-overexpressing ( D ) or -knockdown ( E ) HCC1937 cells were treated as indicated. Data are represented as mean ± SEM, n = 3 biological replicates. P value by paired T -test. F Correlation between RASAL2 expression and in vivo tumour response to doxorubicin. Mice were treated with vehicle control or 2 mg/kg doxorubicin . Each dot represents an independent TNBC PDX model. Tumour growth inhibition was defined as [1 − (mean volume of treated tumours)/(mean volume of control tumours)] × 100%. Pearson r and P value are reported. G Change in tumour volume following doxorubicin in two TNBC PDX models. Mice were treated as described in ( F ), n = 8–9 per group. TM00099 tumours had the lowest RASAL2 expression, whereas TM01278 had the highest RASAL2 expression. P value by two-way ANOVA test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Derivative Assay, Viability Assay, Plasmid Preparation, Control, Knockdown, In Vivo, Inhibition

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: A Immunoblotting of cisplatin-treated TNBC cells showing upregulation of apoptotic/DNA damage markers, cleaved caspase 3 and γH2AX, respectively. Vector control and RASAL2-overexpressing MDA-MB-468 cells were treated with 3 µM cisplatin for 24 h before being released into fresh drug-free medium for the indicated durations. B Immunoblotting showing upregulation of apoptotic marker, cleaved caspase 3, in vector control but not RASAL2-overexpressing MDA-MB-468 cells following apoptosis induction. Cells were treated with either DMSO or 10 µM staurosporine for 1 h. C GSEA analysis of transcriptomes showing downregulation of apoptosis signatures in RASAL2-overexpressing MDA-MB-468 cells compared to isogenic vector cells. D GSEA analysis of transcriptomes showing downregulation of apoptosis signatures in RASAL2-high TNBC tumours versus RASAL2-low TNBC tumours. TNBC patients from the TCGA cohort were stratified by quartile of RASAL2 expression. E Heatmaps of apoptosis-related genes for isogenic RASAL2-overexpressing MDA-MB-468 cells. BCL2 was the topmost significantly upregulated anti-apoptotic gene in RASAL2-overexpressing cells. F Immunoblotting of isogenic TNBC cell lines showing upregulation of BCL2 protein upon RASAL2 expression. G Quantitative immunofluorescence of BCL2 expression in isogenic RASAL2-overexpressing MDA-MB-468 cell model. Data are represented as mean ± SEM. P value by two-tailed T -test. Scale bar, 40 µm. H Representative immunohistochemistry showing TNBC tumours with high RASAL2/BCL2 protein expression (top panel) versus low RASAL2/BCL2 protein expression (bottom panel). Quantification was done based on the H-score system (Fig. S ). Scale bar, 50 µm. I Dot plots showing upregulation of BCL2 mRNA expression in post-treatment primary TNBC/ BRCA -mutant tumours versus pre-treatment tumours. Data are represented as mean ± SEM. P value by two-tailed T -test. J Immunoblotting of isogenic MDA-MB-468 cells following siRNA knockdown of RASAL2 or BCL2. Knocking down RASAL2 downregulated BCL2 expression, while knocking down BCL2 did not change RASAL2 expression.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Western Blot, Plasmid Preparation, Control, Marker, Expressing, Immunofluorescence, Two Tailed Test, Immunohistochemistry, Mutagenesis, Knockdown

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: A Venn diagram showing the overlapped predicted transcription factors binding on the promoters of RASAL2 and BCL2 using computational tools, JASPAR and LASAGNA. B Analyses of candidate transcription factors in breast cancer patient cohorts. Heatmap shows the fold changes of RASAL2, BCL2 and candidate gene transcription factors in patient-matched breast tumour specimens post- versus pre-treatment (top, ). Correlation between RASAL2/BCL2 expression and candidate transcription factors in TNBC patients in the TCGA-BRCA cohort (bottom). P value by two-sided Pearson correlation analysis. C Consensus binding motifs of transcription factor CREB1. D Decrease in the relative mRNA expression of RASAL2 and BCL2 following siRNA-mediated knockdown of CREB1 in MDA-MB-468 cells. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-tailed T -test. E Immunoblotting of TNBC cells transfected with siCREB1 or control siRNA. CREB1, RASAL2 and BCL2 were decreased in expression in cells treated with siCREB1 compared to control. F ChIP-qPCR confirmation of CREB1 binding to predicted sites on RASAL2 and BCL2 promoters. TSS denotes transcription start site. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-tailed T -test. G Decrease in the relative luciferase units in siCREB1 MDA-MB-468 cells compared to siControl. pRL-CMV Renilla luciferase plasmid was co-transfected for normalisation. Data are represented as mean ± SEM, n = 3 biological replicates. P value by two-way ANOVA. H Decrease in the relative luciferase units in MDA-MB-468 cells with truncated RASAL2 promoter without CREB1-binding sequence compared to those with wild-type (WT) RASAL2 promoter. pRL-CMV Renilla luciferase plasmid was co-transfected for normalisation. Data are represented as mean ± SEM, n = 3 biological replicates. P value by one-way ANOVA test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Binding Assay, Expressing, Knockdown, Two Tailed Test, Western Blot, Transfection, Control, ChIP-qPCR, Luciferase, Plasmid Preparation, Sequencing

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: A Immunofluorescence of BCL2 and RASAL2 in primary TNBC patient tumour. Scale bar, 20 µm. B Immunofluorescence of BCL2 and RASAL2 in TNBC cells. Bottom graph shows the line scan quantification of BCL2 (red) and RASAL2 (green). Scale bar, 30 µm. C Confocal imaging of BCL2 and RASAL2 in TNBC cells. Panels on the left show exemplary co-localisation of signals within the boxed region of the cell. Scale bar, 10 µm. D AlphaFold prediction of the interaction between BCL2 and the N-terminus of RASAL2. pLDDT score (0–100) is a confidence score, and pTM score (0–1) is a metric for the structural congruency between two folded protein structures, with higher scores corresponding to higher confidence. PAE plot of the top ranked model is shown on the right [ , ]. E Co-immunoprecipitation of BCL2 and RASAL2 in MDA-MB-468 cells. F Immunoblotting of cytoplasmic versus mitochondrial fractions of mammary cell lines. BCL2 was not detected in 4T1 murine cells as the antibody used was reactive only to human. AKT and TOM20 serve as cytoplasmic and mitochondrial markers, respectively. G Confocal imaging of RASAL2 and MitoTracker in TNBC cells. Scale bar, 5 µm.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Immunofluorescence, Imaging, Immunoprecipitation, Western Blot

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: A Live-cell imaging. RASAL2 depletion increases the rate of GFP-BAX accumulation (green) in HCC1937 cells following exposure to 20 µM staurosporine. Number denotes time in seconds. Scale bar, 10 µm. B Quantification of change in BAX intensity. Fluorescence intensity of individual BAX foci was tracked over time and quantified, n = 4 foci per condition. Data are represented as mean ± SEM. P value by two-tailed T -test. C Schematic for live mitochondrial outer membrane permeabilisation (MOMP) assay. D MOMP assays revealing attenuated cytochrome c release in RASAL2-overexpressing TNBC cells. TOM20 serves as mitochondrial marker. E JC-1 mitochondrial membrane potential assay. TNBC cells were treated with vehicle DMSO or 5 µM doxorubicin (DOXO), and subsequently stained with JC-1 reagent. JC-1 aggregates (indicating high mitochondrial membrane potential) were observed as red, while JC-1 monomers (indicating low mitochondrial membrane potential) were green. Representative images of vector control and RASAL2-overexpressing TNBC cells are shown. Scale bar, 100 µm. F Quantification of the ratio of integrated intensity of red to green fluorescence in ( E ). Data are represented as mean ± SEM, n = 5 random fields of view per condition. P value by two-tailed T -test.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Live Cell Imaging, Fluorescence, Two Tailed Test, Membrane, Marker, Staining, Plasmid Preparation, Control

Journal: Oncogene

Article Title: CREB1-BCL2 drives mitochondrial resilience in RAS GAP-dependent breast cancer chemoresistance

doi: 10.1038/s41388-025-03284-5

Figure Lengend Snippet: RASAL2 and BCL2 share common transcription factor motifs in their promoter regions. Transcription factor CREB1 binds to these promoter regions, and drives the expression of RASAL2 and BCL2. This upregulation is supported by CREB1-interactor YAP, a transcription co-factor that is regulated by RASAL2, thus forming a positive loop in the CREB1-RASAL2-BCL2 axis. Both RASAL2 and BCL2 colocalise at the mitochondria. Their presence confers mitochondrial resilience by mitigating mitochondrial outer membrane depolarisation, which occurs, for example, during BAX/tBID-triggered apoptosis. Consequently, in high RASAL2/BCL2 chemoresistant tumour cells, there is reduced cytochrome c release upon apoptosis induction and thereby attenuation of cell death.

Article Snippet: The full-length wild-type RASAL2 and the N-terminal truncated (lacking nucleotides +1 to +819) variants from cell lysates were then immunoprecipitated using GFP-Trap® agarose beads (ChromoTek).

Techniques: Expressing, Membrane

Journal: The EMBO Journal

Article Title: Splice‐dependent trans‐synaptic PTP δ– IL 1 RAPL 1 interaction regulates synapse formation and non‐ REM sleep

doi: 10.15252/embj.2019104150

Figure Lengend Snippet: A Schematic diagram for the dissection of SR and SLM layers in the hippocampus (P21–27). B–F Representative immunoblots of total lysates from the SR and SLM layers for the tested synaptic proteins, including proteins known to be enriched in the SLM (NGL‐1 and HCN1), the PTPδ relative PTPσ, postsynaptic partners of PTPδ (IL1RAPL1, Slitrk2/3, and NGL‐3), presynaptic scaffolds/adaptors (Bassoon and liprin‐α), postsynaptic scaffolds/adaptors (CaMKIIα/β, PSD‐95, SynGAP1), postsynaptic receptors (GluA1/2, GluN1, GluN2A/B), and signaling molecules (phospho‐Src). α‐Tubulin was used as a control ( n = 4 mice for WT and KO, mean ± SEM, *** P < 0.001, ns, not significant, two‐way ANOVA, Tukey's HSD post‐hoc test). G Normal synaptic levels of other PTPδ‐binding partners (Slitrk3, SALM3, and NGL‐3) in the Ptprd −/− brain (P21–27), as revealed by immunoblotting of crude synaptosomal (P2), synaptic plasma membrane (SPM), and PSD (PSD II) fractions ( n = 3 mice [WT and KO] for each fraction [P2, SPM, and PSD], mean ± SEM, ns, not significant, Student's t ‐test). H Normal synaptic levels of other PTPδ‐binding partners (Slitrk3, SALM3, and NGL‐3) in the Ptprd‐meA −/− brain (P21–27), as revealed by immunoblotting of P2, SPM, and PSD (PSD II) fractions ( n = 3 mice [ Ptprd‐meA +/+ and Ptprd‐meA −/− ] for each fraction [P2, SPM, and PSD], mean ± SEM, ns, not significant, Student's t ‐test). Source data are available online for this figure.

Article Snippet: The following antibodies were previously described: NGL‐1 (#2040) (Um et al , ), NGL‐3 (#1948) (Lee et al , ), GluA1(#1193), GluA2 (#1195) (Kim et al , ), liprin‐α (#1289) (Ko et al , ), SynGAP1 (#1682) (Kim et al , ).The following antibodies were commercially purchased: GluN2A (Alomone AGC‐003), GluN2B (Neuromab 75‐101), IL1RAPL1 (Proteintech, 21609‐1‐AP), IL1RAcP (Millipore, ABT333), Slitrk2 (Abcam ab67305), Slitrk3 (Abcam ab67306), mCherry (Abcam, ab125096), pTyr (4G10) (Millipore, 05‐321), HCN1 (Neuromab 75‐110), Bassoon (Stressgene, VAM‐PS003), p‐Src (Cell Signaling, 2105), PSD‐95 (Neuromab 75‐028), α‐tubulin (Sigma T5168), β‐actin (Sigma A5316), CaMKIIα/β (Cell Signaling 3362).

Techniques: Dissection, Western Blot, Binding Assay