Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Virus infection induces delayed STAT1 Ser-708 phosphorylation. A, WT or IKKϵ−/− MEFs were mock-stimulated or stimulated with 100IU/ml IFN-β. Protein lysate was collected 16 h post-IFN stimulation and immunoblotted using p-STAT1 Ser-708, p-STAT1 Ser-727, p-STAT1 Tyr-701, and total STAT1 antibodies. B and C, Sendai and WNV virus infections induce STAT1 Ser-708 phosphorylation. HEK293 cells were infected with 100 HA units/ml SenV (B) or West Nile virus strain Madagascar (WNV-MAD) (C) at an m.o.i. of 1. At the indicated times following infection, protein lysates were collected and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, p-IRF-3, total IRF-3, IFIT1, and SenV or WNV. Tubulin and GAPDH were used as loading controls.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Type I, type II, and type III IFNs induce STAT1 Ser-708 phosphorylation. 2fTGH cells were mock-treated or treated with 100 IU/ml IFN-β (A) or 50 ng/ml IFN-γ (B). C, PH5CH8 cells were mock-treated (lane 1), treated with 100 ng/ml IFN-λ1 (lanes 2-6), or 100 IU/ml IFN-β (lanes 7-9). Protein lysate was collected at respective time points following IFN treatment and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Signaling through IFNAR is required for STAT1 Ser-708 phosphorylation following type I IFN treatment or virus infection. WT, IRF-3−/−, or IFNAR−/− (A) and parental 2fTGH cells or their derivative U5A cells (which lack IFNAR) (B) were infected with WNV-MAD at an m.o.i. of 1. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, and WNV. C and D, the same cells were also mock-stimulated or stimulated with 100 IU/ml IFN-β for 6 or 16 h. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, IFIT2, IFIT3, and IFIT1. Asterisk, nonspecific band.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: STAT1 Ser-708 phosphorylation requires de novo protein synthesis, STAT1 tyrosine dephosphorylation, and nuclear export. A, 2fTGH cells were mock-treated (-CHX, lanes 1-5) or treated with CHX (+CHX, lanes 6-11) to block protein synthesis. At 30 min (lanes 6-10) or 16 h (lane 11) following CHX treatment, cells were mock-stimulated (M) or stimulated with IFN-β. Cells were harvested at 10 min as well as 1, 6, and 16 h post-IFN stimulation and immunoblotted to detect p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, ISG15, and IFIT1. B, 2fTGH cells were not treated (NT, lanes 1-3), pretreated with 100 nm LMB (lanes 4-6), or 50 mm pervanadate (Van, lanes 7-9) 1 h before mock stimulation or stimulation with 100 IU/ml IFN-β. Cells were harvested at 1 and 16 h post-stimulation. Immunoblot analysis was performed using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1 antibodies.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: De-Phosphorylation Assay, Blocking Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: IKKϵ mediates IFIT2 expression and protection against WNV pathogenesis in vivo. WT Bl6 and IKKϵ−/− mice were mock-infected (PBS only) or infected with 103 pfu of WNV-MAD subcutaneously through footpad injection. A, mice were monitored and scored daily for clinical symptoms over 17 days. Clinical scores from four representative mice per group were graphed. B, spleens from WT or IKKϵ−/− mice, mock-infected or infected with WNV-MAD, were collected at days 4, 6, and 12 post-infection. Protein lysates were extracted by homogenizing spleens with radioimmune precipitation assay buffer and immunoblotted using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, IFIT2, IFIT1, WNV, and IKKϵ antibodies. The immunoblot analysis panel is a representative from four mice per infection group.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Expressing, In Vivo, Infection, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: A model illustrating that early and late ISGs induction is regulated by multiple STAT1 posttranslational modifications. 1, the canonical JAK-STAT signaling is activated following type I IFN binding to its receptor, which results in STAT1 Tyr-701 phosphorylation, ISGF3 formation, and its nuclear translocation. ISGF3 binding to the ISRE element induces transcription of ISGs. 2, chromatin-bound STAT1 can be phosphorylated by MAPK at residue Ser-727, which induces its sumoylation. 3 and 4, nuclear STAT1 is also acetylated by histone acetyltransferase (HAT) CREB-binding protein (CBP), resulting in recruitment of TCP1, which catalyzes STAT1 tyrosine dephosphorylation. Sumoylated-acetylated STAT1 cycles back to the cytoplasm, and both modifications render STAT1 unable to be further tyrosine-phosphorylated. 5 and 6, type I IFN signaling and unknown IFN-stimulated factor(s) activate IKKϵ phosphorylation of STAT1 Ser-708. 7, STAT1 molecules phosphorylated at Ser-708 can enter the nucleus and induce expression of a specific ISG subset. pY, tyrosine phosphorylation; pS, serine phosphorylation; Ac, acetylation; Su, sumoylation.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Binding Assay, Translocation Assay, De-Phosphorylation Assay, Expressing

Journal: Frontiers in Insect Science

Article Title: Protein localization of aquaporins in the adult female disease vector mosquito, Aedes aegypti

doi: 10.3389/finsc.2024.1365651

Figure Lengend Snippet: Sizes and functional characteristics of aquaporins examined in the current study found in Aedes aegypti along with their orthologs in the African malaria vector mosquito, Anopheles gambiae , and the fruit fly, Drosophila melanogaster .

Article Snippet: WB tissue sections were probed with one of the following; Anti-AaAQP1 affinity-purified primary antibody (1:1000 rabbit polyclonal antibody against CFFKVRKGDEESYDF, Genscript, NJ, USA) , Anti-AaAQP2 affinity purified primary antibody (1:50 rabbit polyclonal antibody against CNGLGNTGLKENVQD, Genscript, NJ, USA) , Anti-AaAQP4 affinity purified primary antibody (1:500 rabbit polyclonal antibody against PAEQAPSDVGKSNQS, Genscript, NJ, USA) , Anti-AaAQP5 affinity purified primary antibody (1:1000 rabbit polyclonal antibody against FRREVPEPEYNRELT, Genscript, NJ, USA) , or Anti-AaAQP6 affinity purified primary antibody (1:50 rabbit polyclonal antibody against CSFRNMFLADKAKAE, Genscript, NJ, USA).

Techniques: Functional Assay, Plasmid Preparation

Journal: Frontiers in Insect Science

Article Title: Protein localization of aquaporins in the adult female disease vector mosquito, Aedes aegypti

doi: 10.3389/finsc.2024.1365651

Figure Lengend Snippet: Localization of AaAQP1, 2, and 6 in whole body adult female Ae. aegypti. Immunohistochemical localization of water selective AaAQPs (red staining) in female Ae. aegypti mosquitoes (~10–12 days old). Each AaAQP (red staining) was localized under non-blood fed conditions, 0.5hr post blood meal conditions, and 24hr post blood meal conditions. Whole body sections of the abdominal segment immunolocalizing AaAQP1 in (A–C) , AaAQP2 in (D–F) , and AaAQP6 in (G–I) . In (B) , an inset image (B’) shows AaAQP1 membrane localization in the HG. In (E) , an inset image (E’) shows AaAQP2 membrane localization in the MG. All images were taken at 10x magnification (n=3). The Na + /K + -ATPase (NKA) was used as a membrane marker (green staining) and nuclei were stained with DAPI (blue). MT, Malpighian tubules; FB, fat body; OV, ovaries; HG, hindgut; MG, midgut; RP, rectal pads. White arrows indicate aggregated staining of AaAQP in the MTs.

Article Snippet: WB tissue sections were probed with one of the following; Anti-AaAQP1 affinity-purified primary antibody (1:1000 rabbit polyclonal antibody against CFFKVRKGDEESYDF, Genscript, NJ, USA) , Anti-AaAQP2 affinity purified primary antibody (1:50 rabbit polyclonal antibody against CNGLGNTGLKENVQD, Genscript, NJ, USA) , Anti-AaAQP4 affinity purified primary antibody (1:500 rabbit polyclonal antibody against PAEQAPSDVGKSNQS, Genscript, NJ, USA) , Anti-AaAQP5 affinity purified primary antibody (1:1000 rabbit polyclonal antibody against FRREVPEPEYNRELT, Genscript, NJ, USA) , or Anti-AaAQP6 affinity purified primary antibody (1:50 rabbit polyclonal antibody against CSFRNMFLADKAKAE, Genscript, NJ, USA).

Techniques: Immunohistochemical staining, Staining, Membrane, Marker

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 3.2 ( a , c ) or 5.2 ( b ) pmol Control-MO (Control) or FOLR1-MO (FOLR1 KD) per embryo and incubated with saline or 50 μM pteroate until the neural tube closed in control embryos, when they were fixed, photomicrographed ( a , b ), sectioned and processed for immunostaining ( c ). a , b Examples of embryos in each group. Arrowheads indicate open neural tube (neural tube defect, NTD). Numbers are embryos presenting open (NTD, purple) or closed (green) neural tube. Bar graphs represent proportion of phenotypes in each group. c Examples of immunostained transverse sections of the neural tissue; n of embryos sectioned was 25, 39 and 42 in Control, FOLR1 KD, and FOLR1 KD+pteroate groups, respectively, in N = 3 independent experiments. Scale bar is 20 μm. Graph shows distribution of neural tissue defect score per group, median is indicated by dashed line. In ( a – c ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA, Tukey post-hoc multiple comparison test ( a , b ), or Kruskal-Wallis, Dunn’s multiple comparison test ( c ). Source data are provided as a Source Data file.

Article Snippet: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

Techniques: Control, Incubation, Saline, Immunostaining, Comparison

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 1.6 pmol Control-MO (Control) or FOLR1-MO (FOLR1 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. 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 = 42 and 44 neural plates for Control and FOLR1 KD groups, respectively, N = 8 independent experiments. b – e Neural plate stage Xenopus laevis embryos were fixed, sectioned and processed for immunostaining. b – d Examples of immunostained neural plate. Arrows indicate colocalization of C-cadherin with early endosomal marker (EEA1) and ubiquitin ( b ), late endosomal marker (Rab7, c ) and lysosomal marker (LAMP1, d ). Scale bar, 10 μm. e Graph shows mean ± SD proportion of C-cadherin vesicles colocalizing with the indicated markers. Number of cells analyzed were 104, 52, 65 from immunostained samples in ( b , c and d ), respectively, from N = 5 embryos. f Two-cell stage Xenopus laevis embryos were bilaterally microinjected with hEEA1-GFP mRNA and membrane mCherry and unilaterally microinjected with 2.6–3.2 pmol FOLR1-MO (FOLR1 KD) or Control-MO (Control) 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. Schematic shows experimental design. Xenopus illustrations © Natalya Zahn (2022) obtained from Xenbase ( www.xenbase.org RRID:SCR_003280), released under a Creative Commons Attribution-NonCommercial 4.0 License (CC BY-NC) . Images are maximum intensity projection of single time frame. Dashed lines indicate border between wild-type (WT) and MO-injected neural plate. Insets show neural plate injected side with 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; ns: not significant, n = 30 cells analyzed in each group, N = 5 embryos per group. Scale bar, 20 μm. In a , f , * p < 0.05, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

Techniques: Control, Western Blot, Two Tailed Test, Immunostaining, Marker, Ubiquitin Proteomics, Membrane, Injection, Labeling

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 bilaterally microinjected with 1.6 pmol Control-MO (Control) or FOLR1-MO (FOLR1 KD) per blastomere, incubated from early neural plate stage (stage 12) with vehicle or proteasome and lysosome inhibitors until they reached mid-neural plate stages (stage 16–17) when neural plates were dissected and processed for Western blot ( a , b ) and immunoprecipitation ( b ) assays. a Example of Western blot assay immunoprobed for C-cadherin showing full-length and cleaved (~80 kD) forms. Graph shows individual and mean ± SD percent of optical density (OD) for cleaved C-cadherin band normalized with GAPDH protein band OD and compared to controls, n = 20 and 26 neural plates for Control and FOLR1 KD groups respectively, N = 3 independent experiments. b Example of immunoprecipitation (IP) assay for C-cadherin followed by Western blot assay for ubiquitin and C-cadherin. Graph shows individual and mean ± SD percent of optical density (OD) for ubiquitinated (Ubiq) C-cadherin band normalized with full-length C-cadherin and compared to controls. In ( a , b ), * p < 0.05, ** p < 0.01, two-tailed ratio t -test, n = 16 and 24 neural plates for Control and FOLR1 KD groups respectively, N = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

Techniques: Control, Incubation, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, 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 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: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

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: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

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: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

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: IP/Western blot antibodies were rabbit anti- Xenopus laevis CD2AP polyclonal affinity purified antibody raised against the peptide CRPKSEVEPHSKTKT custom made by GenScript and anti- Xenopus laevis FOLR1 antibody (GenScript).

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