Journal: Nature communications

Article Title: The Dishevelled-associating protein Daple controls the non-canonical Wnt/Rac pathway and cell motility.

doi: 10.1038/ncomms1861

Figure Lengend Snippet: Figure 2 | Daple induces Rac activation through its binding with Dvl. (a) The activities of Rac in the lysates from HEK293T cells transfected with either control (empty), full-length Daple or CT domain of Daple (Daple CT) were assessed by GST-PAK-PBD pull-down assays. The precipitates and total cell lysates (TCL) were analysed with the indicated antibodies (upper panel) and quantified (lower panel). The results represent the mean values ± s.d. of three experiments. Significant difference was determined by Student’s t-test. *P < 0.05, as compared with control cells. (b) Schematic illustration of Daple CT tagged with the V5 epitope and its mutant Daple CT∆GCV that lacked the binding site for Dvl. (c) Lysates from HEK293T cells transfected with either Daple CT or Daple CT∆GCV were immunoprecipitated with control IgG or anti-V5 antibody. The precipitates and the TCL were analysed by western blot, using the indicated antibodies. (d) The activities of Rac in lysates from HEK293T cells transfected with either Daple CT or Daple CT∆GCV were assessed by GST-PAK-PBD pull-down assays. (e) The activities of Rac in lysates from HEK293T cells transfected with the indicated combinations of plasmids (control or Daple CT) and siRNAs (control or Dvl2 siRNA) were assessed by GST-PAK-PBD pull-down assays. Daple-CT-induced Rac activation was cancelled by the knockdown of endogenous Dvl2. (f) The activities of Rac in the lysates from HEK293T cells transfected with the indicated combinations of plasmids (control or HA-Dvl2) and siRNAs (control or Daple siRNA) were assessed by GST-PAK-PBD pull-down assays. The precipitates and the TCL were analysed by western blot using the indicated antibodies.

Article Snippet: Anti-myc and anti-Dvl2 (10B5) antibodies for western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Binding Assay, Transfection, Control, Mutagenesis, Immunoprecipitation, Western Blot, Knockdown

Journal: Nature communications

Article Title: The Dishevelled-associating protein Daple controls the non-canonical Wnt/Rac pathway and cell motility.

doi: 10.1038/ncomms1861

Figure Lengend Snippet: Figure 3 | Daple regulates the interaction between Dvl and PKC in the activation of Rac. (a, b) Lysates from HEK293T cells transfected with control (empty), Daple CT or Daple CT∆GCV were immunoprecipitated with either anti-Dvl2 (a) or anti-PKCλ (b) antibody. The precipitates and the total cell lysates (TCL) were analysed by western blot, using the indicated antibodies. (c) Lysates from HEK293T cells transfected with either control or Daple siRNA were immunoprecipitated with either anti-PKCλ antibody or normal mouse IgG. The precipitates and the TCL were analysed by western blot using the indicated antibodies. (d) Monolayers of confluent HEK293T cells that had been transfected with either control or Daple siRNA were serum-starved for 24 h and stimulated with Wnt5a (100 ng ml − 1). The monolayers were wounded and incubated for an additional 30 min. TCL were immunoprecipitated with Dvl2 antibody, followed by western blot analysis using the indicated antibodies. (e) Lysates from HEK293T cells transfected with myc-PKCλ, and either GST, GST-Daple CT, or GST-Daple CT∆GCV were precipitated with glutathione-Sepharose beads. The precipitates and the TCL were analysed by western blot, using the indicated antibodies. (f) HEK293T cells were transfected with either control plasmid or Daple CT, and were incubated in the absence or presence of the aPKC inhibitor, cell-permeable myristoylated aPKC pseudosubstrate, (20 µM) for 1 h. Rac activation was monitored by GST-PAK-PBD pull-down assays. The precipitates and the TCL were analysed by western blot, using the indicated antibodies. (g) HEK293T cells were serum-starved and incubated with Wnt5a either in the absence or presence of the aPKC inhibitor, followed by GST-PAK-PBD pull-down assays to monitor Rac activation. (h) TCL prepared from HEK293T cells transfected with control vector or Daple CT were analysed by western blot, using the indicated antibodies.

Article Snippet: Anti-myc and anti-Dvl2 (10B5) antibodies for western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Control, Immunoprecipitation, Western Blot, Incubation, Plasmid Preparation

Journal: Nature communications

Article Title: The Dishevelled-associating protein Daple controls the non-canonical Wnt/Rac pathway and cell motility.

doi: 10.1038/ncomms1861

Figure Lengend Snippet: Figure 4 | Daple/Dvl interaction regulates actin remodelling. (a, b) Expression of Daple and Daple CT, but not Daple CT∆GCV, induced lamellipodia formation in Vero cells cultured in growth medium containing 10% FBS. Arrowheads indicate the lamellipodia (a). The number of cells with lamellipodia was counted in each group and quantified (b). (c) Vero cells transfected with green fluorescent protein (GFP)–Daple were fixed and stained with the indicated antibodies (left panel). The region within the white box is shown at a higher magnification in the right upper panel. Pixel intensities of the green (GFP), red (β-actin) and blue (CMAC) channels were quantified in the sections indicated by Lines 1 and 2 in the right upper panel. The plots indicate pixel intensity (y axis) over the 15–30 µm sections (x axis; distance in µm). (d) Vero cells cultured in growth medium containing 10% FBS were transfected with either control or Daple siRNA. The basal activity of Rac was monitored with a GST-PAK-PBD pull-down assay. (e, f) Vero cells transfected with either control or Daple siRNA were fixed and stained with the indicated antibodies. The regions within the white boxes are shown at a higher magnification in adjacent panels. Shown in (f) is the quantification of lamellipodia observed in control and Daple knockdown cells. (g, h) HA-Dvl2-transfected cells were fixed and stained with the indicated antibodies. The regions within the boxes are magnified in the lower panels. Shown in (h) is the quantification of localisation of Daple and Dvl2 at the leading edge. Pixel intensities of the green (Daple), red (Dvl2) and blue (CMAC) channels were quantified in 20-µm sections indicated by an arrow in the left lower panel. (i) Myc-Daple-transfected Vero cells were fixed and stained with the indicated antibodies. For the staining of endogenous Dvls, a cocktail of antibodies containing anti-Dvl2 and anti-Dvl3 was used. The regions within the white boxes are shown at a higher magnification in lower panels. All bars represent the mean values ± s.d. of three experiments. Significant difference was determined by Student’s t-test. *P < 0.05, as compared with control cells. All scale bars, 20 µm.

Article Snippet: Anti-myc and anti-Dvl2 (10B5) antibodies for western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Cell Culture, Transfection, Staining, Control, Activity Assay, Pull Down Assay, Knockdown

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 2 | DVL1 knockdown reduces β-catenin levels in the cartilage and chondrocytes. Mice with OA were administered adenoviral vectors containing either sh-DVL1 or sh-NC via intra-articular injections. Lentiviral vectors carrying sh-DVL1 or sh-NC were introduced into chondrocytes extracted from OA mice. The mRNA (A) and protein (B) levels of DVL1 in chondrocytes were determined using RT-qPCR and WB analysis, respec- tively. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) The transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Positive staining of DVL1 in the mouse knee joint determined using immunofluorescence staining. (F) mRNA expression of DVL1 in the joint cartilage determined using RT-qPCR. (G) Positive expression of β-catenin in the joint cartilage determined us- ing IHC assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C, D) or ANOVA (A, B, F, and G).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Knockdown, Quantitative RT-PCR, Activity Assay, Staining, Immunofluorescence, Expressing

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 3 | DVL1 knockdown ameliorates cartilage injury in mice. (A) Protein levels of COL10A1, MMP13, and SOX9 in mouse cartilage tissues determined using WB analysis. (B) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (C) Positive TRAP staining in the mouse knee joints. (D) Apoptosis in the extracted chondrocytes determined using TUNEL assay. (E) Protein levels of pro- cleaved-caspase-3 in the extracted chondrocytes determined using WB analysis. (F) Protein levels of COL10A1, MMP13, and SOX9 in the mouse chondrocytes determined using WB analysis. For animal experiments, each group contained five mice. For cell experiments, three biological repli- cates were performed. Differences were compared by the unpaired t-test (D–F) or ANOVA (A–C).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Knockdown, Staining, TUNEL Assay

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 4 | CHIR-99021 restores cartilage injury mitigated by DVL1 silencing. Mice stably administered sh-DVL1 were further treated with the Wnt/β-catenin agonist CHIR-99021 via intra-articular injection. (A) Protein levels of β-catenin in cells determined using WB analysis. (B) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (C) Protein levels of COL10A1 and COL2A1 in mouse chon- drocytes determined using WB analysis. (D) Positive TRAP staining in the mouse knee joints; in vitro, chondrocytes with stable DVL1 knockdown were treated with 5 μM CHIR-99021 for 24 h. (E) Apoptosis in the extracted chondrocytes determined using TUNEL assay. (F) Protein levels of pro- cleaved-caspase-3 in the extracted chondrocytes determined using WB analysis. (G) Protein levels of expression of MMP13 and SOX9 in the mouse chondrocytes determined using WB analysis. For animal experiments, each group contained five mice. For cell experiments, three biological repli- cates were performed. Differences were compared by the unpaired t-test (A–G).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Stable Transfection, Injection, Staining, In Vitro, Knockdown, TUNEL Assay, Expressing

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 5 | SP2, poorly expressed in OA, represses DVL1 transcription. (A) Transcription factor binding sites near the mouse DVL1 promoter region predicted using the JASPAR Transcription Factors plugin from the UCSC Genome Browser. (B) Intersections of the predicted transcription factors and the significant DEGs obtained from high-throughput sequencing. (C) Positive staining of SP2 in the joint cartilage determined using IHC. (D) SP2 mRNA expression in the joint cartilage determined using RT-qPCR. (E) Binding between SP2 and the DVL1 promoter in chondrocytes determined using ChIP-qPCR assay; the extracted chondrocytes were administered lentiviral vectors carrying OE-NC or OE-SP2. (F) mRNA and (G) protein level of SP2 in mouse chondrocytes determined using RT-qPCR and WB analysis. (H) mRNA expression of DVL1 in mouse chondrocytes analyzed using RT-qPCR. (I) Regulation of SP2 on transcription activity of the DVL1 promoter in chondrocytes determined using the dual lucifer- ase reporter gene assay. For animal experiments, each group contained five mice. For cell experiments, three biological replicates were performed. Differences were compared by the unpaired t-test (C–I).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Binding Assay, Next-Generation Sequencing, Staining, Expressing, Quantitative RT-PCR, ChIP-qPCR, Activity Assay, Reporter Gene Assay

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 6 | DVL1 upregulation aggravates cartilage injury in mice alleviated by SP2. DMM-challenged mice were introduced with adenoviral vectors carrying OE-NC/OE-SP2 alone or with the additional OE-NC/OE-DVL1. (A) mRNA expression of SP2 and DVL1 in the joint cartilage deter- mined using RT-qPCR. (B) Positive staining of SP2 and DVL1 in the joint cartilage determined using IHC. (C) Protein levels of β-catenin in the joint cartilage determined using WB analysis. (D) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (E) Positive TRAP staining of the mouse knee joints. (F) Protein levels of COL10A1 and MMP13 in the mouse cartilage determined using WB analysis. Each group contained five mice. Differences were compared by ANOVA (A–F).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 7 | DVL1 overexpression increases chondrocyte loss suppressed by SP2. Chondrocytes were administered lentiviral vectors encapsu- lating OE-NC/OE-SP2 or the additional OE-NC/OE-DVL1. (A) mRNA expression of DVL1 in cells determined using RT-qPCR. (B) Apoptosis in cells determined using TUNEL assay. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) Transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Protein levels of SOX9 and COL2A1 in the chondrocytes determined using WB analysis. Three biological replicates were performed. Differences were compared by unpaired t-test (A) or ANOVA (B–E).

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, TUNEL Assay, Activity Assay

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 8 | Schematic illustration of the mechanism. In cartilage tissues of osteoarthritic mice, decreased expression of SP2 resulted in its attenuated transcriptional repression of DVL1. Aberrantly expressed DVL1 activated the β-catenin signaling, leading to chondrocyte damage and osteoarthritis progression.

Article Snippet: The sections were blocked with 5% goat serum for 1 h, followed by an overnight incubation at 4°C with the antibodies against DVL1 (1:100, 13–706, ProSci, Poway, California, United States), β- catenin (1:500, ab32572, Abcam), and SP2 (1:100, PA5- 103254, Thermo Fisher Scientific).

Techniques: Expressing

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: YTHDF1 and YTHDF2 regulate local translation of Dvl1 and Wnt5a , respectively, to control the GC axon growth. A) Relative Dvl1 protein level detected by TMT‐labeled proteomic analysis after YTHDF1 KD. Data are mean ± SEM: * p = 0.047; n = 3 replicates; by unpaired Student's t test. B) RT‐qPCR confirming the Dvl1 mRNA level was unchanged after KD of YTHDF1 in GCs. Data are mean ± SEM: p = 0.78; n = 3; ns, not significant; by unpaired Student's t test. C) Relative Wnt5a mRNA level measured by RNA‐seq after YTHDF2 KD. Data are mean ± SEM: **** p = 4.92E‐05; n = 3 replicates; by unpaired Student's t test. D) Axon growth rate significantly increased after KD of Dvl1. Quantification of axon growth rates after KD of Dvl1 using siRNAs. Data are represented as box and whisker plots: n = 21 axons for each group; siDvl1#4 versus siCtrl , **** p = 2.09E‐05; siDvl1#5 versus siCtrl , **** p = 3.15E‐10. All by one‐way ANOVA followed by Tukey's multiple comparison test. E) Axon growth rate significantly decreased after KD of Wnt5a. Quantification of axon growth rates after KD of Wnt5a using siRNAs. Data are represented as box and whisker plots: n = 20 axons for each group; siWnt5a#1 versus siCtrl , **** p = 4.79E‐05; siWnt5a#3 versus siCtrl , **** p = 4.96E‐06. All by one‐way ANOVA followed by Tukey's multiple comparison test. F) Dvl1 and Wnt5a mRNAs were detected in axons by RT‐PCR using total RNA from pure axons or soma, respectively. Similar to β‐actin mRNA which is a positive control for axonal mRNAs, Dvl1 and Wnt5a mRNAs were detected in both axons and soma. The absence of H1f0 mRNA from axons indicated that the axonal material was pure with no soma incorporation. G,H) Detection of Dvl1 and Wnt5a mRNA localization in growth cones of GC neurons by FISH. Dissociated GCs were cultured for 2 DIV and then FISH was performed using RNAscope riboprobes. Dvl1 and Wnt5a mRNAs were detected in growth cones of GC neurons as red punctate patterns. β‐actin and Dapb serve as positive and negative controls, respectively. Tuj1 immunostaining was used to visualize axons. Quantification of puncta density was shown in (H). I,J) Compartmentalized KD of Dvl1 in GC axons. GCs were cultured in microfluidic chambers and siDvl1 was specifically transfected to axons only. Compared with siCtrl, siDvl1#4 and siDvl1#5 led to significant decrease of Dvl1 IF signals. Quantification data are represented as box and whisker plots (J). siDvl1#4 ( n = 15 axons) versus siCtrl ( n = 18 axons), **** p = 1.11E‐11; siDvl1#5 ( n = 17 axons) versus siCtrl , **** p = 3.56E‐12; by one‐way ANOVA followed by Tukey's multiple comparison test. K) Axon growth rates significantly increased after axon‐specific KD of Dvl1. Data are represented as box and whisker plots. siDvl1#4 ( n = 18 axons) versus siCtrl ( n = 21 axons), ** p = 0.0024; siDvl1#5 ( n = 17 axons) versus siCtrl , *** p = 0.00049; by one‐way ANOVA followed by Tukey's multiple comparison test. L,M) Compartmentalized KD of Wnt5a in axons. Compared with siCtrl , siWnt5a#1 and siWnt5a#3 led to significant decrease of Wnt5a IF signals. Quantification data are represented as box and whisker plots (M). siWnt5a#1 ( n = 38 axons) versus siCtrl ( n = 32 axons), **** p = 6.80E‐14; siWnt5a#3 ( n = 39 axons) versus siCtrl , **** p = 5.20E‐14; by one‐way ANOVA followed by Tukey's multiple comparison test. N) Axon growth rates significantly decreased after axon‐specific KD of Wnt5a which can be rescued by application of recombinant Wnt5a protein into axonal compartments. Data are represented as box and whisker plots. siWnt5a#1 ( n = 15 axons) versus siCtrl ( n = 16 axons), **** p = 3.86E‐06; siWnt5a#3 ( n = 19 axons) versus siCtrl , **** p = 1.59E‐07; siWnt5a#1 +rWnt5a ( n = 18 axons) versus siCtrl , p = 0.34; siWnt5a#3 +rWnt5a ( n = 16 axons) versus siCtrl , p = 0.85; siWnt5a#1 +rWnt5a versus siWnt5a#1 , * p = 0.036; siWnt5a#3 +rWnt5a versus siWnt5a#3 , **** p = 3.40E‐05; ns, not significant; by one‐way ANOVA followed by Tukey's multiple comparison test. O) Overexpression of YTHDF1 increased axonal Dvl1 protein level in cultured GCs and axon‐specific siDvl1 KD eliminated this increase. Data are represented as box and whisker plots. Ythdf1‐IRES‐GFP + siCtrl versus IRES‐GFP + siCtrl , **** p = 1.34E‐05; Ythdf1‐IRES‐GFP + siDvl1#4 versus IRES‐GFP + siDvl1#4 , p = 0.99; Ythdf1‐IRES‐GFP + siDvl1#5 versus IRES‐GFP + siDvl1#5 , p = 0.84; IRES‐GFP + siDvl1#4 versus IRES‐GFP + siCtrl , p = 5.60E‐14; IRES‐GFP + siDvl1#5 versus IRES‐GFP + siCtrl , p = 5.80E‐14; Ythdf1‐IRES‐GFP + siDvl1#4 versus Ythdf1‐IRES‐GFP + siCtrl , **** p = 1.01E‐15; Ythdf1‐IRES‐GFP + siDvl1#5 versus Ythdf1‐IRES‐GFP + siCtrl , **** p = 1.02E‐15; ns, not significant; n = 27 axons for each group; by one‐way ANOVA followed by Tukey's multiple comparison test. P) KD of YTHDF2 increased axonal Wnt5a protein level in GCs and axon‐specific siWnt5a KD eliminated this increase. Data are represented as box and whisker plots. shYthdf2#3 + siCtrl versus shCtrl + siCtrl , **** p = 4.30E‐15; shYthdf2#3 + siWnt5a#1 versus shCtrl + siWnt5a#1 , p = 0.99; shYthdf2#3 + siWnt5a#3 versus shCtrl + siWnt5a#3 , p = 0.89; shCtrl + siWnt5a#1 versus shCtrl + siCtrl , **** p = 1.34E‐11; shCtrl + siWnt5a#3 versus shCtrl + siCtrl , p = 1.01E‐13; shYthdf2#3 + siWnt5a#1 versus shYthdf2#3 + siCtrl , **** p = 1.01E‐15; shYthdf2#3 + siWnt5a#3 versus shYthdf2#3 + siCtrl , **** p = 1.02E‐15; ns, not significant; n = 27 axons for each group; by one‐way ANOVA followed by Tukey's multiple comparison test. Scale bars represent G) 10 µm and I,L) 5 µm.

Article Snippet: Sources and dilutions of antibodies used in WB are as follows: YTHDF1 (17479‐1‐AP, Proteintech) 1:2500; YTHDF2 (24744‐1‐AP, Proteintech) 1:2500; Dvl1 (27384‐1‐AP, Proteintech) 1:1000; Wnt5a (55184‐1‐AP, Proteintech) 1:1000; HA (ab18181, Abcam) 1:2000; Tau1 (MAB3420, Millipore) 1:1000; Tag1 (AF1714, R&D Systems) 1:2500; GluR δ 2(sc‐26118, Santa Cruz Biotechnology) 1:1000; Nrxn1 (ab77596, Abcam) 1:1000; GAPDH (10494‐1‐AP, Proteintech) 1:1000; β ‐actin (ab6276, Abcam) 1:30 000; β ‐actin (AC004, Abclonal) 1:30 000; Donkey anti‐goat IgG H&L (HRP) (ab97110, Abcam) 1:2500; Donkey anti‐mouse IgG H&L (HRP) (ab97030, Abcam) 1:2500; Donkey anti‐rabbit IgG H&L (HRP) (ab16284, Abcam) 1:2500; VHH anti‐mouse IgG secondary antibody (HRP) (KTSM1321, AlpaLife) 1:5000; VHH anti‐rabbit IgG secondary antibody (HRP) (KTSM1322, AlpaLife) 1:5000.

Techniques: Control, Labeling, Quantitative RT-PCR, RNA Sequencing, Whisker Assay, Comparison, Reverse Transcription Polymerase Chain Reaction, Positive Control, Cell Culture, RNAscope, Immunostaining, Transfection, Recombinant, Over Expression

Journal: Advanced Science

Article Title: The m 6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

doi: 10.1002/advs.202101329

Figure Lengend Snippet: A working model shows that YTHDF1 and YTHDF2 work synergistically to regulate Wnt5a‐PCP signaling pathway and cerebellar granule cell axon growth. A) Under normal conditions, YTHDF1 promotes the translation of m 6 A‐modified Dvl1 mRNA in GC axons. Dvl1 can block Wnt5a‐Fzd3‐activated PCP signaling. Meanwhile, YTHDF2 facilitates Wnt5a mRNA degradation to downregulate Wnt5a protein level in GC axons. So both YTHDF1 and YTHDF2 negatively regulate Wnt5a‐PCP signaling pathway and GC axon growth. B) In Ythdf1 and Ythdf2 cKO mice, local translation of Dvl1 mRNA and decay of Wnt5a mRNA in GC axons are inhibited, respectively. The resulting downregulation of Dvl1 and upregulation of Wnt5a protein levels in axons potentiate Wnt5a‐PCP signaling and promote GC axon growth.

Article Snippet: Sources and dilutions of antibodies used in WB are as follows: YTHDF1 (17479‐1‐AP, Proteintech) 1:2500; YTHDF2 (24744‐1‐AP, Proteintech) 1:2500; Dvl1 (27384‐1‐AP, Proteintech) 1:1000; Wnt5a (55184‐1‐AP, Proteintech) 1:1000; HA (ab18181, Abcam) 1:2000; Tau1 (MAB3420, Millipore) 1:1000; Tag1 (AF1714, R&D Systems) 1:2500; GluR δ 2(sc‐26118, Santa Cruz Biotechnology) 1:1000; Nrxn1 (ab77596, Abcam) 1:1000; GAPDH (10494‐1‐AP, Proteintech) 1:1000; β ‐actin (ab6276, Abcam) 1:30 000; β ‐actin (AC004, Abclonal) 1:30 000; Donkey anti‐goat IgG H&L (HRP) (ab97110, Abcam) 1:2500; Donkey anti‐mouse IgG H&L (HRP) (ab97030, Abcam) 1:2500; Donkey anti‐rabbit IgG H&L (HRP) (ab16284, Abcam) 1:2500; VHH anti‐mouse IgG secondary antibody (HRP) (KTSM1321, AlpaLife) 1:5000; VHH anti‐rabbit IgG secondary antibody (HRP) (KTSM1322, AlpaLife) 1:5000.

Techniques: Modification, Blocking Assay