Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Proportion of PB integration sites across the genome: promoters (15.13%), untranslated region (UTR) (1.04%), exons (2.09%), introns (32.12%), and intergenic regions (49.64%). ( B ) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the top 200 genes with the most frequent insertions from the S-iTSCs. VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor. ( C ) The top 10 candidate genes among the inserted genes from the S-iTSCs. ( D ) Sense (red) and antisense (green) insertions of PB in Catip and Dyrk1a gene bodies from the S-iTSCs. The rectangles indicate the exons. ( E ) Schematic diagram of the strategy to Catip -KO and Dyrk1a -KO in WT-ESCs by using CRISPR-Cas9 system. ( F ) Sequences of genotypes in the Catip -KO and Dyrk1a -KO ESCs.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: CRISPR

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) The live cell numbers of the Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs during daily culture. Catip -KO and Dyrk1a -KO did not affect the proliferation of ESCs. n.s., not significant. ( B ) Chromosome spread analysis of Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs (diploid). Scale bars, 10 μm. ( C ) Statistical analysis of chromosome numbers in the Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs. ( D ) Teratomas formed from the Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs were identified by hematoxylin and eosin staining. Scale bars, 100 μm. The tissues shown were glands (endoderm), blood (mesoderm), and neural tissue (ectoderm). ( E ) GO analysis of DEGs between Catip -KO ESCs and WT-ESCs. ( F ) GO analysis of DEGs between Dyrk1a -KO ESCs and WT-ESCs. Pol II, polymerase II.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: Staining

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Phase-contrast images of established Catip -KO iTSCs and Dyrk1a -KO iTSCs. Scale bars, 100 μm. ( B ) Immunofluorescence of TSC-specific markers CDX2 (green) and EOMES (green) in the Catip -KO iTSCs and Dyrk1a -KO iTSCs. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( C ) Immunofluorescence of the trophoblast lineage–specific markers PROLIFERIN (green), TPBPA (green), and SDC1 (green) in differentiated cells derived from the Catip -KO iTSCs and Dyrk1a -KO iTSCs. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( D ) PCA of RNA-seq datasets from the WT-ESCs, Catip -KO ESCs, Dyrk1a -KO ESCs, Cdx2 -OE ESCs, WT-TSCs, Catip -KO iTSCs, Dyrk1a -KO iTSCs, and Cdx2 -OE iTSCs. ( E ) Genome browser tracks showing CpG methylation status in Pou5f1 and Elf5 loci of WT-ESCs, Catip -KO ESCs, Dyrk1a -KO ESCs, WT-TSCs, Catip -KO iTSCs, and Dyrk1a -KO iTSCs. ( F ) Bubble plot representing the expression of Catip , Dyrk1a , Cdx2 , Zscan4b , Dux , and Oct4 in early embryonic developing stages and ESCs. ( G ) Schematic diagram of the DYRK1A inhibitor–harmine. ( H ) Phase-contrast images of established harmine + iTSCs and WT-TSCs. Scale bars, 100 μm.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: Immunofluorescence, Staining, Derivative Assay, RNA Sequencing, CpG Methylation Assay, Expressing

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Heatmap showing some DEGs between Dyrk1a -KO ESCs and WT-ESCs mapping to Notch pathway genes, Hippo/Yap pathway genes, and totipotent genes. ( B ) PCA results of RNA-seq datasets from the WT-ESCs, Dyrk1a -KO ESCs, TBLCs, TLSCs, and ciTotiSCs. ( C ) GSEA of DEGs associated with totipotent genes in Dyrk1a -KO ESCs. NES, normalized enrichment score; FDR, false discovery rate. ( D ) The Venn diagram of the up-regulated DEGs between Dyrk1a -KO ESCs and TBLCs, versus WT-ESCs. ( E ) Top 10 genes in the co–up-regulated DEGs between Dyrk1a -KO ESCs and TBLCs, versus WT-ESCs. ( F ) GO analysis of co–up-regulated DEGs between Dyrk1a -KO ESCs and TBLCs, versus WT-ESCs.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: RNA Sequencing

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Immunofluorescence of CDX2 (red) in chimeric blastocyst from GFP-labeled Dyrk1a -KO ESCs and WT-ESCs. Donor cells were GFP + (green), and DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( B ) Images of E12.5 chimeras (including fetus, placenta, and yolk sac) derived from GFP-labeled Dyrk1a -KO ESCs, with uninjected embryo as a control. Scale bars, 5 mm. ( C ) FACS analysis of GFP + cells in the fetus, placenta, and yolk sac in E12.5 chimeras derived from the GFP-labeled Dyrk1a -KO ESCs. The percentages of GFP + cells in the fetus, placenta, and yolk sac of Dyrk1a -KO chimera were 43.6, 14.9, and 54.4%, respectively; uninjected ones (fetus, placenta, and yolk sac) at E12.5 were as a control. ( D ) Immunofluorescence staining of placental-specific markers (TPBPA, PROLIFERIN, TFAP2C, and GCM1, each in red) in the GFP-labeled Dyrk1a -KO ESC–derived E12.5 placentas. The white arrows indicated that GFP-positive cells could contribute to the tissues expressing specific markers. Scale bars, 75 μm. ( E ) Phase-contrast images of blastoid derived from the Dyrk1a -KO ESCs. WT-blastocyst was used as a control. Scale bars, 10 μm. ( F ) Immunofluorescence of CDX2 (green) and OCT4 (red) in blastoid derived from the Dyrk1a -KO ESCs. WT-blastocyst was used as a control. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( G ) Immunofluorescence of CDX2 (green) and GATA6 (red) in blastoid derived from the Dyrk1a -KO ESCs. WT-blastocyst was used as a control. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: Immunofluorescence, Labeling, Staining, Derivative Assay, Control, Expressing

Journal: Cell Death & Disease

Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

doi: 10.1038/s41419-024-06828-x

Figure Lengend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

Article Snippet: F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis.

Techniques: Flow Cytometry, Expressing, RNA Sequencing, ChIP-qPCR, Binding Assay, Negative Control, Activity Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased tau-induced inflammatory responses are associated with a greater degree of atherosclerosis in progressive supranuclear palsy

doi: 10.3389/fnagi.2025.1608631

Figure Lengend Snippet: Greater atherosclerotic burden and elevated plasma total tau, p-tau181 levels in patients with PSP. Using carotid artery Doppler ultrasound to assess the degree of carotid artery atherosclerosis by evaluating (A) max-CIMT, (B) max-CPT, (C) TPN, (D) CPS, and (E) carotid stenosis% in HCs ( n = 56) and patients with PSP ( n = 56). Using MRA to evaluate the degree of intracranial arterial atherosclerosis by recording (F) ICS vascular territory and (G) MRA GSS in HCs ( n = 56) and patients with PSP ( n = 56). (H) Heatmap of multivariable linear regression heatmap showing the association between disease duration/clinical scores H&Y staging scale, UPDRS-III, PSPRS, MMSE and atherosclerosis measures (max-CIMT, max-CPT, TPN, CPS, carotid stenosis% and MRA GSS) in patients with PSP ( n = 56). Peripheral (I–L) blood lipid, (M) glycosylated hemoglobin, (N) homocysteine parameters, and plasma levels of (O) total tau, (P) p-tau181, (Q) p-tau396 in HCs ( n = 56) and patients with PSP ( n = 56). (R) Heatmap of multivariable linear regression heatmap showing the association between tau levels (total tau, p-tau181, p-tau396) and atherosclerosis measures (max-CIMT, max-CPT, TPN, CPS, carotid stenosis% and MRA GSS) in patients with PSP ( n = 56). Results are expressed as mean and SD. Statistical approaches: (A–E, G, I–Q) covariance (ANCOVA), with age, gender, smoking status and stroke history as covariates, (F) chi-square test, (H, R) multivariable linear regression. In heatmaps, each cell represents the standardized regression coefficient (β) derived from multivariable linear regression models, where each Y -axis was regressed on X -axis while adjusting for age, gender, smoking history, hypertension, diabetes, and dyslipidemia (For MMSE, one more correction for period of education). The color intensity of heatmap indicates the strength and direction of association (β), with positive associations shown in red and negative in blue. P < 0.05 indicates statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001), and ns indicates p ≥ 0.05.

Article Snippet: The following ELISA kits were used: Human interleukin-6 (IL-6) ELISA kit (E-HSEL-H0003, Elabscience Biotechnology, Wuhan, China), Human interleukin-1β (IL-1β) ELISA kit (E-HSEL-H0001, Elabscience Biotechnology, Wuhan, China), Human interleukin-10 (IL-10) ELISA kit (E-EL-H0005, Elabscience Biotechnology, Wuhan, China), Human interferon γ (IFN-γ) ELISA kit (E-HSEL-H0007, Elabscience Biotechnology, Wuhan, China), Human tumor necrosis factor-α (TNF-α) ELISA kit (E-HSEL-H0109c, Elabscience Biotechnology, Wuhan, China), Human total tau ELISA kit (ml057755, mlbio, Shanghai, China),and Human P-tau181 ELISA kit (ml057691, mlbio, Shanghai, China), Human P-tau396 ELISA kit (E-EL-H5314c, Elabscience Biotechnology, Wuhan, China), Mouse interleukin-6 (IL-6) ELISA kit (ml098430, mlbio, Shanghai, China), Mouse interleukin-1β (IL-1β) ELISA kit (ml098416, mlbio, Shanghai, China) and Mouse tumor necrosis factor-α (TNF-α) ELISA kit (mIC50536-1, mlbio, Shanghai, China).

Techniques: Clinical Proteomics, Derivative Assay

Journal: Frontiers in Aging Neuroscience

Article Title: Increased tau-induced inflammatory responses are associated with a greater degree of atherosclerosis in progressive supranuclear palsy

doi: 10.3389/fnagi.2025.1608631

Figure Lengend Snippet: Elevated peripheral blood macrophage ratio and plasma inflammatory cytokine levels in patients with PSP. Representative flow cytometry analysis of (A) macrophages (CD14 + CD68+), and (B) classically activated macrophages (CD86 + CD206-M1) subpopulations from peripheral blood (left) and quantifications (right) in HCs ( n = 10) and patients with PSP ( n = 10). Plasma levels of inflammatory cytokine (C) CRP, (D) IL-6, (E) IL-1β, (F) IL-10, (G) TNF-α, and (H) IFN-γ in HCs ( n = 56) and PSP with patients ( n = 56). (I) Heatmap of multivariable linear regression heatmap showing the association between inflammatory cytokine levels (CRP, IL-6, IL-1β, IL-10, TNF-α, and IFN-r) and atherosclerosis measures (max-CIMT, max-CPT, TPN, CPS, carotid stenosis% and MRA GSS) in patients with PSP ( n = 56). (J) Heatmap of multivariable linear regression heatmap showing the association between tau levels (total tau, p-tau181, p-tau396) and inflammatory cytokine levels (CRP, IL-6, IL-1β, IL-10, TNF-α, and IFN-r) in patients with PSP ( n = 56). Results are expressed as mean and SD. Statistical approaches: (A, B) unpaired non-parametric Mann-Whitney U tests, (C–H) covariance (ANCOVA), with age, gender, smoking status and stroke history as covariates, (I, J) multivariable linear regression. In heatmaps, each cell represents the standardized regression coefficient (β) derived from multivariable linear regression models, where each Y -axis was regressed on X -axis while adjusting for age, gender, smoking history, hypertension, diabetes, and dyslipidemia. The color intensity of heatmap indicates the strength and direction of association (β), with positive associations shown in red and negative in blue. P < 0.05 indicates statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001), and ns indicates p ≥ 0.05.

Article Snippet: The following ELISA kits were used: Human interleukin-6 (IL-6) ELISA kit (E-HSEL-H0003, Elabscience Biotechnology, Wuhan, China), Human interleukin-1β (IL-1β) ELISA kit (E-HSEL-H0001, Elabscience Biotechnology, Wuhan, China), Human interleukin-10 (IL-10) ELISA kit (E-EL-H0005, Elabscience Biotechnology, Wuhan, China), Human interferon γ (IFN-γ) ELISA kit (E-HSEL-H0007, Elabscience Biotechnology, Wuhan, China), Human tumor necrosis factor-α (TNF-α) ELISA kit (E-HSEL-H0109c, Elabscience Biotechnology, Wuhan, China), Human total tau ELISA kit (ml057755, mlbio, Shanghai, China),and Human P-tau181 ELISA kit (ml057691, mlbio, Shanghai, China), Human P-tau396 ELISA kit (E-EL-H5314c, Elabscience Biotechnology, Wuhan, China), Mouse interleukin-6 (IL-6) ELISA kit (ml098430, mlbio, Shanghai, China), Mouse interleukin-1β (IL-1β) ELISA kit (ml098416, mlbio, Shanghai, China) and Mouse tumor necrosis factor-α (TNF-α) ELISA kit (mIC50536-1, mlbio, Shanghai, China).

Techniques: Clinical Proteomics, Flow Cytometry, MANN-WHITNEY, Derivative Assay

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Neural plate stage Xenopus laevis embryos were processed for co-immunoprecipitation (IP) assays. Example of Western blot assay from immunoprecipitates with FOLR1 or GFP (control) antibodies and probed for CD2AP or FOLR1. Similar results were observed in N = 3 independent experiments. b Neural plate stage Xenopus laevis embryos were fixed and processed for immunostaining. Images are transverse single z-sections of immunostained neural plate showing apical colocalization of phospho-CD2AP (p-CD2AP) and p-c-Cbl with C-cadherin. Scale bar, 10 μm. Similar results were observed in N = 3 independent experiments. c Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 9.9 pmol of Control-morpholino (Control, Control-MO), 2.6 pmol CD2AP-MO1 (CD2AP KD1) or 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD2/KD) per blastomere until neural tube closed in control embryos, when they were fixed and photomicrographed. Examples of whole embryos in each group. Arrowheads indicate open neural tube (neural tube defect, NTD). Numbers are embryos presenting closed (green) or open (purple, NTD) neural tube. Bar graph represents proportion of phenotypes in each group. d Two-cell stage Xenopus laevis embryos were unilaterally microinjected with 9.9 pmol Control-MO (Control) and 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD) along with GFP and mCherry mRNA, respectively, and allowed to develop until they reached mid-neural plate stages, when they were fixed and processed for immunostaining. Image is a transverse section of the neural plate, immunostained for GFP (Control), mCherry (CD2AP KD) and C-cadherin. Double arrows indicate apical surface length of medial superficial Control (white) and CD2AP KD (magenta) neural plate cells. Scale bar, 20 μm. Graph shows individual and mean ± SD apical length of superficial neural plate cells per embryo, n of cells = 75 in each half of the neural plate, N of embryos = 4. **** p < 0.0001, two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Immunoprecipitation, Western Blot, Control, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Two-cell stage Xenopus laevis embryos were bilaterally microinjected with hEEA1-GFP and membrane mCherry mRNAs and unilaterally microinjected with 9.9 pmol CD2AP-MO (CD2AP KD) per blastomere along with fluorescent tracer and allowed to develop until they reached early neural plate stages (stage 13-14), when they were time-lapse imaged with an acquisition rate of 1 frame/6 min. Image is maximum intensity projection of single time frame. Dashed line indicates border between morpholino-injected and wild-type (WT) neural plate. Inset shows neural plate injected side showing tracer in blue. Graphs show distribution between both halves of the neural plate (in %) of the number of EEA1-GFP vesicles and area fraction of labeled endosomes per neural plate cell surface. Two-tailed paired t -test, n = 28 cells analyzed in each group from N = 5 embryos per group. Scale bar, 20 μm. b Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 7.4 pmol Control-MO (Control) or CD2AP-MO (CD2AP KD) per blastomere and allowed to grow until they reached mid-neural plate stages (stage 15–17) when neural plate was dissected and processed for Western blot assays. Image is an example of Western blot assay. Graph shows individual and mean ± SD percent of optical density (OD) for C-cadherin immunoblot band normalized with GAPDH protein band OD and compared to controls. Two-tailed ratio t -test, n = 28 and 24 neural plates for Control and CD2AP KD groups, respectively, N = 5 independent experiments. In ( a , b ), * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Membrane, Injection, Labeling, Two Tailed Test, Control, Western Blot

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: Two-cell stage Xenopus laevis embryos were microinjected with 14.8 pmol Control-morpholino (MO, Control, a , b ), 3.2 pmol FOLR1-MO (FOLR1 KD, a ) or 14.8 pmol CD2AP-MO2 (CD2AP KD, b ) per embryo and incubated with saline or proteasome and lysosome inhibitors at the end of gastrulation (stage 12) until neural plate stages (stage 17) when they were processed for Western blot assays. Images are examples of Western blot assays. Graphs show individual and mean ± SD percent of optical density (OD) for CD2AP ( a ) or FOLR1 ( b ) immunoblot band normalized with GAPDH protein band OD and compared to controls. In ( a ), n = 34 and 42 neural plates for Control and FOLR1 KD, respectively, N = 7 independent experiments; n = 20 and 26 neural plates for Control+inhibitors and FOLR1 KD+inhibitors groups, respectively, N = 3 independent experiments. In ( b ) n = 16 and 20 for Control and CD2AP KD groups, respectively and n = 16 and 18 neural plates for Control+inhibitors and CD2AP KD+inhibitors, respectively, N = 3 independent experiments. In ( a , b ) * p < 0.05, *** p < 0.001, ns: not significant, two-tailed ratio t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Control, Incubation, Saline, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a – c Neural plate from mid neural plate stage Xenopus laevis embryos was dissected and dissociated cells plated in vitro. After 2 h, cells were loaded with the Ca 2+ sensor Fluo4-AM and time-lapse imaged. a Example of 1-h recording of neural plate cell Ca 2+ activity. b , c Folinic acid ( b , c ), folic acid ( c ) or vehicle was added to neural plate cells in culture during time-lapse imaging and the Ca 2+ response was recorded in the first minute post addition. b Example of acute transient elicited by 100 μM folinic acid. c Graph shows mean ± SEM folinic- or folic acid-responsive neural plate cells compared to total number of cells with spontaneous Ca 2+ transients in 30 min recording, N = 3 independent experiments. Two-tailed one sample t and Wilcoxon test. d Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA and grown until early and mid-neural plate stages when they were time-lapse imaged before and after addition of vehicle or 300 μM folinic acid. Image shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles. Graph shows individual and mean ± SD percent change in Ca 2+ transient frequency before and after addition of vehicle or folinic acid, n = 4, 5, 6 and 7 embryos for Early-Vehicle, Early-Folinic acid, Mid-Vehicle and Mid-Folinic acid groups, respectively. One-sample two-tailed t -test, compared to hypothetical value of 100%. e – h Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA ( e – h ) and unilaterally microinjected with 9.9 pmol FOLR1-MO1 (FOLR1 KD1/KD) or 1.6 pmol FOLR1-MO2 (FOLR1 KD2) per blastomere ( e ) and grown until mid-neural plate stages when they were time-lapse imaged. Image in ( e ) shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles in WT and FOLR1 KD1 halves. Graphs show individual Ca 2+ transient frequency (transients/5 min) in WT and FOLR1 KD1 or KD2 halves ( e , n = 6 embryos) and in WT embryos before and after addition of vehicle ( f , n = 7 embryos), 50 μM folic acid ( g , n = 6 embryos), Na + and voltage-gated Ca 2+ channel blockers (VGC block : 0.02% tricaine+10 μM nitrendipine+25 μM TTA-2, h , n = 5 embryos), or a mix of folic acid and VGC block ( h , n = 5 embryos). Two-tailed paired t -test ( e – g ) and 1-way ANOVA-Tukey multiple comparisons test ( h ). In ( c – h ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. i Model of FOLR1 and CD2AP regulation of neural tube formation. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: In Vitro, Activity Assay, Imaging, Two Tailed Test, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

doi: 10.1074/jbc.m609990200

Figure Lengend Snippet: FIGURE 1. ZIP kinase kinase activities in smooth muscle extracts. Smooth muscle extracts were enriched for protein kinase activity by -phosphate ATP affinity chromatography and then fractionated by microanion-exchange chromatography. In A, chromatography column fractions were assayed for protein kinase activity with the indicated peptides derived from the regula- tory phosphorylation sites of ZIPK and MYPT1 in the presence and absence of 10 M Y-27632. B shows the respective activities of recombinant ZIPK and ROCK1 against a peptide derived from the sequence surrounding Thr-265 on ZIPK. C shows the respective activities of purified recombinant ZIPK and ROCK1 against peptides containing the regulatory phosphorylation sites on MYPT1.

Article Snippet: Phosphospecific antibodies were prepared from phosphosynthetic peptides derived from the Thr-180, Thr-225, Thr-265, Thr-299, Thr-306, and Ser-311 phosphorylation sites of ZIPK by Proteintech Inc. All other antibodies were purchased from Calbiochem.

Techniques: Activity Assay, Affinity Chromatography, Chromatography, Derivative Assay, Phospho-proteomics, Recombinant, Sequencing, Purification

Journal: Journal of Biological Chemistry

Article Title: ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

doi: 10.1074/jbc.m609990200

Figure Lengend Snippet: FIGURE 3. Characterization of the primary ROCK1 phosphorylation sites on ZIPK. The D161A mutant form of full-length recombinant ZIPK was phosphorylated to 1.4 mol/mol with purified ROCK1. A shows an auto- radiogram of the phosphorylated protein; recombinant ROCK1 was identified in the upper band, and recom- binant ZIPK was identified in the lower band. In B the ZIPK band was digested with trypsin, and the 32P-labeled phosphopeptides were characterized by reverse phase HPLC and Cerenkov counting of column fractions. In C isolated phosphopeptides were characterized by CRP analysis as described under “Experimental Procedures.” In D and E 32P-labeled ZIPK was digested with lysyl endopeptidase C and characterized by reverse phase HPLC and CRP analysis as described in Fig. 1, B and C.

Article Snippet: Phosphospecific antibodies were prepared from phosphosynthetic peptides derived from the Thr-180, Thr-225, Thr-265, Thr-299, Thr-306, and Ser-311 phosphorylation sites of ZIPK by Proteintech Inc. All other antibodies were purchased from Calbiochem.

Techniques: Phospho-proteomics, Mutagenesis, Recombinant, Purification, Labeling, Isolation

Journal: Journal of Biological Chemistry

Article Title: ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

doi: 10.1074/jbc.m609990200

Figure Lengend Snippet: FIGURE 4. ROCK1 phosphorylates and activates ZIPK in vitro. In A, a series of phosphospecific antibodies were derived to the indicated phosphoryla- tion sites on ZIPK. The antibodies were tested for specificity against auto- phosphorylated ZIPK and recombinant ZIPK with the indicated Ser/Thr 3 Ala substitutions. In B phosphorylation of Thr-265 and Thr-299 was measured in vitro following phosphorylation of D161A ZIPK with increasing concentra- tions of ROCK1. In C, the activity of wild type (WT), T225A, and T265A was measured against the MYPT1T696 peptide following preincubation with the indicated amounts of ROCK. The preincubation assay was halted after 10 min by addition of 10 M Y-27632.

Article Snippet: Phosphospecific antibodies were prepared from phosphosynthetic peptides derived from the Thr-180, Thr-225, Thr-265, Thr-299, Thr-306, and Ser-311 phosphorylation sites of ZIPK by Proteintech Inc. All other antibodies were purchased from Calbiochem.

Techniques: In Vitro, Derivative Assay, Recombinant, Phospho-proteomics, Activity Assay