Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Representative RT-PCR showing that mRNA for PKD1 is present in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl but absent in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl :Cdh5(PAC)-CreERT2 mice. Representative of 5 independent experiments for each genotype. (B) Representative Western blots illustrating PKD1, PKD2, AT1, eNOS, Fzd-7 and actin proteins in mesenteric arteries of Pkd1 fl/fl and Pkd1 ecKO mice. (C) Mean data from experiments shown in panel B. Significance was assessed using Student t-tests, n=5 independent mesenteric arterial lysates for each genotype and each protein. (D) Immunofluorescence images (representative of three mesenteric arteries from three mice for each genotype) illustrating that PKD1 (Alexa Fluor 546) is abolished in endothelial cells of en face mesenteric arteries from Pkd1 ecKO mice. CD31 (Alexa Fluor 488) and DAPI are also shown. Scale bars, 50 μm. (E) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt9b (3 μg/ml) applied in continuous flow in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (F) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt9b (3 µg/ml) applied under continuous flow (10 dyn/cm 2 ) in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (G) Mean data from experiments shown in panels E and F, and the modulation of flow and Wnt9b-mediated vasodilation by SRI37892 (Fzd-7 Inh, 5 µM). Significance was assessed using a two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=10 arteries from 7 mice of each genotype for flow, flow+ Wnt9b and Wnt9b. n=7 arteries from 5 mice for Fzd-7 Inh+flow and Fzd-7 Inh+flow+Wnt9b. n=5 arteries from 5 mice for each genotype for flow + boiled Wnt9b. (H) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt5a (3 µg/ml). (I) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt5a (3 µg/ml). (J) Mean data from experiments shown in panels H and I and the modulation of flow and Wnt5a-induced vasodilation by SRI37892 (Fzd-7 Inh, 5 μM). Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=8 arteries from 6 mice from Pkd1 fl/fl for flow, flow+ Wnt5a. n=5 arteries from 5 mice from Pkd1 ecKO for flow, flow+ Wnt5a. n=5 arteries from 5 mice of each genotype for flow + boiled Wnt5a. n=8 arteries from 6 mice from Pkd1 fl/fl for flow+ Fzd-7 Inh and flow+ Wnt5a+ Fzd-7 Inh.

Article Snippet: Membranes were blocked with 5% milk or 5% BSA and incubated with one of the following primary antibodies: PKD1 (Polycystic Kidney Disease Research Resource Consortium, Baltimore), PKD2 (Alomone), eNOS (Abcam), p-eNOS (Cell Signaling), Wnt9b (R&D Systems), Wnt5a (R&D Systems), AT1 receptor (Alomone) or actin (Cell Signaling) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, Immunofluorescence

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Wnt3a or Wnt5a specifically reduced the cell surface levels of V5-FZD5/8. HEK293A cells stably expressing each of the 10 V5-FZDs (FZD1 to 10) were treated with control, Wnt3a, or Wnt5a CM for 4 hours, and the cell surface levels of V5-FZDs were analyzed via flow cytometry using an anti-V5 antibody. ( B ) Wnt3a or Wnt5a specifically decreased the levels of mature forms of V5-FZD5/8. The whole cell lysates (WCLs) from the indicated cells treated as described in ( A ) were analyzed by immunoblotting with the indicated antibodies. ( C ) Bafilomycin A1 (BA1) restored Wnt3a or Wnt5a induced FZD5 degradation. ( D ) Schematic diagram of FZD5 constructs: full-length FZD5, FZD5 lacking the extracellular cysteine-rich domain (FZD5△CRD), and FZD5 lacking the intracellular C-terminus (FZD5△C). ( E , F ) HEK293A cells stably expressing the FZD5 truncation constructs shown in ( D ) were treated and analyzed as described in ( A ). ( G ) Schematic diagram of CRD-swapped chimeric FZDs. ( H ) HEK293A cells stably expressing the indicated chimeric FZD constructs shown in ( F ) were treated and analyzed as described in ( A ). Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Stable Transfection, Expressing, Control, Flow Cytometry, Western Blot, Construct, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Wnt3a or Wnt5a specifically reduced the cell surface levels of V5-FZD5/8. Huh7 cells stably expressing each of the 10 V5-FZDs (FZD1 to 10) were treated with control, Wnt3a, or Wnt5a CM for 4 hr, and the cell surface levels of V5-FZDs were analyzed via flow cytometry using an anti-V5 antibody. ( B ) Wnt3a or Wnt5a specifically decreased the levels of mature forms of V5-FZD5. The whole cell lysates (WCLs) from the indicated U2OS cells treated as described in ( A ) were analyzed by immunoblotting with the indicated antibodies. Figure 1—figure supplement 1—source data 1. Raw unedited blots for . Figure 1—figure supplement 1—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Stable Transfection, Expressing, Control, Flow Cytometry, Western Blot, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Wnt3a or Wnt5a induced the degradation of endogenous FZD5. HEK293A cells with a V5 epitope tag knocked in at the C-terminus of endogenous FZD5 (FZD5KI) were treated with Wnt3a or Wnt5a CM for the indicated durations, and the WCLs were analyzed by immunoblotting with the indicated antibodies. Wild-type (WT) cells served as a negative control to confirm the specificity of the FZD5KI bands. The upper bands represent mature FZD5, whereas the lower bands represent immature FZD5. ( B ) Wnt3a or Wnt5a induced the degradation of endogenous FZD5 but not FZD7. FZD5KI or FZD7KI (generated as described for FZD5KI) HEK293A cells were treated with control, Wnt3a CM, or Wnt5a CM for 2 hr, and the WCLs were subjected to immunoblotting with the indicated antibodies. ( C ) The Porcupine inhibitor IWP-2 increased the level of the endogenous mature form of FZD5 but not FZD7. FZD5KI or FZD7KI cells were treated overnight with or without IWP-2 (2.5 μM), and the WCLs were subjected to immunoblotting with the indicated antibodies. ( D, E ) IWP-2 increased the cell surface levels of endogenous FZD5/8 but not FZD4. HEK293A WT cells were treated with or without IWP-2 and analyzed by flow cytometry to determine the FZD5/8 and FZD4 levels on the cell surface with anti-FZD5/8 (2919) or anti-FZD4 (5028) monoclonal antibodies. APC, allophycocyanin. ( F, G ) ZNRF3/RNF43 double knockout increased the cell surface levels of endogenous FZD5/8 but not FZD4. The cell surface levels of FZD5/8 and FZD4 in WT or ZRDKO cells were analyzed by flow cytometry. ( H ) Wnt-induced FZD5 degradation is dependent on endogenous ZNRF3 and RNF43. WT, FZD5KI, or ZRDKO-FZD5KI ( ZNRF3/RNF43 double knockout cells with a V5 tag knocked in at the C-terminus of endogenous FZD5) cells were treated and analyzed as described in ( B ). ( I ) IWP-2 did not increase the endogenous FZD5 protein level in ZRDKO cells. ( J ) RSPO1 treatment increased the level of the mature form of endogenous FZD5 but not FZD7. FZD5KI and FZD7KI cells were treated with control or RSPO1 CM for 4 hr, and the WCLs were analyzed by immunoblotting with the indicated antibodies. ( K ) RSPO1 failed to increase the level of the mature form of FZD5 in ZRDKO cells. FZD5KI or ZRDKO-FZD5KI cells were treated and tested as described in ( J ). ( L ) RSPO1 treatment restored Wnt3a and Wnt5a induced membrane FZD5 degradation. HEK293A FZD5KI cells were incubated with EZ-link Sulfo-NHS-SS-Biotin to label membrane protein, then treated with control, Wnt3a, Wnt5a CM, or Wnt3a/Wnt5a with RSPO1 CM. Membrane proteins were bound by NeutrAvidin beads and eluted for immunoblotting. ( M ) IWP-2 and RSPO1 treatments similarly increased the FZD5/8 levels on the cell surface. WT cells were treated with IWP-2 overnight or RSPO1 for 4 hr, followed by flow cytometry analysis. ( N, O ) RSPO1 treatment did not increase the cell surface levels of FZD5/8 in ZRDKO cells or FZD4 in WT cells. HEK293A ZRDKO or WT cells were treated with control or RSPO1 CM for 4 hr and subjected to flow cytometry analysis with anti-FZD5/8 or anti-FZD4 monoclonal antibodies. Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Western Blot, Negative Control, Generated, Control, Flow Cytometry, Bioprocessing, Double Knockout, Membrane, Incubation, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Validation of HEK293A DVLTKO cells by immunoblotting analysis with the indicated anti-DVL antibodies. ( B ) Triple knockout of DVL1/2/3 increased FZD5/8 levels on the cell surface. WT and DVLTKO cells were analyzed by flow cytometry with an anti-FZD5/8 monoclonal antibody. ( C, D ) IWP-2 ( C ) or RSPO1 ( D ) treatment increased FZD5/8 levels on the cell surface of DVLTKO cells. DVLTKO cells were treated with IWP-2 overnight or RSPO1 for 4 hr, followed by flow cytometry analysis. ( E ) Wnt3a and Wnt5a treatment reduced the cell surface levels of V5-FZD5 in DVLTKO cells. HEK293A DVLTKO cells stably expressing V5-FZD5 were treated with control, Wnt3a CM, or Wnt5a CM for 4 hr and analyzed via flow cytometry with an anti-V5 antibody. ( F ) Wnt3a and Wnt5a treatment decreased the mature form of V5-FZD5 in DVLTKO cells. The cells in ( E ) were treated, and the WCLs were analyzed by immunoblotting with the indicated antibodies. ( G, I ) Triple knockout of DVL1/2/3 reduced ligand-independent FZD5 and FZD7 endocytosis, but had no effect on Wnt3a or Wnt5a induced FZD5 endocytosis. And DVLTKO cells re-expressing DVL2 rescued decreased FZD5 and FZD7 endocytosis caused by DVL1/2/3 triple knockout. HEK293A cells stably expressing V5-linker-FZD5 or V5-linker-FZD7 were first incubated with an anti-V5 antibody, and the cells were treated with control, Wnt3a, or Wnt5a CM for 1 hr and subjected to immunofluorescence analysis. The cells were treated with IWP-2 overnight prior to treatment with conditioned medium. Scale bars, 10 μm. ( H, J ) Quantification of the number of FZD5 ( G ) or FZD7 ( I ) puncta in WT, DVLTKO, and DVLTKO + DVL2 cells (mean ± SD, n=20 cells per group). ns, no significant difference; ***p<0.001; ****p<0.0001. ( K ) WCLs from WT, DVLTKO, and DVLTKO + DVL2 cells stably expressing V5-linker-FZD5 or V5-linker-FZD7 were analyzed by immunoblotting. Figure 3—source data 1. Raw unedited blots for . Figure 3—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Biomarker Discovery, Western Blot, Triple Knockout, Flow Cytometry, Stable Transfection, Expressing, Control, Incubation, Immunofluorescence, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Wnt3a and Wnt5a induced V5-FZD5 endocytosis in ZRDKO cells. ( B ) Wnt3a and Wnt5a induced V5-FZD5 degradation in WT but not ZRDKO cells. ( C ) Wnt5a induced FZD5 internalization in both WT and ZRDKO cells, and internalized FZD5 gradually diminished in WT but not ZRDKO cells. WT or ZRDKO cells stably expressing V5-FZD5 were treated with control or Wnt5a CM for the indicated times and analyzed by immunostaining. The cells were treated with IWP-2 overnight prior to treatment with CM. Scale bars, 10 μm. ( D ) Cotreatment with RSPO1 prevented FZD5 degradation but had little effect on FZD5 internalization induced by Wnt5a. The cells were treated with IWP-2 overnight prior to treatment with CM. Scale bars, 10 μm. ( E ) Compared with those in WT cells, fewer V5-FZD5 puncta colocalized with the early endosomal marker EEA1 in ZRDKO cells. WT or ZRDKO cells stably expressing V5-FZD5 were treated with control or Wnt5a CM for 2 hr and then analyzed by immunostaining. The cells were treated with IWP-2 overnight prior to treatment with CM. Scale bars, 10 μm. ( F ) Quantification of the percentage of V5-FZD5 puncta colocalized with EEA1 in WT and ZRDKO cells (mean ± SD, n=20 cells per group). ****p<0.0001. ( G ) Compared with those in WT cells, fewer V5-FZD5 puncta colocalized with the lysosomal marker LAMP1 in ZRDKO cells. WT or ZRDKO cells stably expressing V5-FZD5 were treated with control or Wnt5a CM for 2 hr and then analyzed by immunostaining. The cells were treated with IWP-2 overnight prior to treatment with CM. Scale bars, 10 μm. ( H ) Quantification of the percentage of V5-FZD5 puncta colocalized with LAMP1 in WT and ZRDKO cells (mean ± SD, n=20 cells per group). ****p<0.0001. Figure 4—source data 1. Raw unedited blots for . Figure 4—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Stable Transfection, Expressing, Control, Immunostaining, Marker, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Wnt3a induced FZD5 internalization in both WT and ZRDKO cells, and internalized FZD5 gradually diminished in WT but not ZRDKO cells. WT or ZRDKO cells stably expressing V5-FZD5 were treated with control or Wnt3a CM for the indicated times and analyzed by immunostaining. The cells were treated with IWP-2 overnight prior to treatment with CM. Scale bars, 10 μm. ( B ) Cotreatment with RSPO1 prevented FZD5 degradation but had little effect on FZD5 internalization induced by Wnt3a. The cells were treated with IWP-2 overnight prior to treatment with CM. Scale bars, 10 μm.

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Stable Transfection, Expressing, Control, Immunostaining

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Flow cytometry analysis of FZD5/8 levels on the cell surface in ZRDKO and ZRDKO-FZD5/8DKO cells with an anti-FZD5/8 antibody. ( B ) Depletion of FZD5/8 diminished the increase in cytosolic β-catenin levels in ZRDKO cells. Cytosolic fractions from the indicated cells were analyzed by immunoblotting. ( C ) Re-expression of V5-FZD5, but not V5-FZD7, restored cytosolic β-catenin levels in ZRDKO-FZD5/8DKO cells. ( D ) Overexpression of V5-FZD, but not V5-FZD7, further elevated cytosolic β-catenin levels in ZRDKO cells. ( E, F ) FZD5, but not FZD7, enhanced the inhibitory effect of RNF43 on Wnt3a signaling. HEK293A cells stably expressing RNF43-HA alone or with V5-FZD5 or V5-FZD7 were treated with increasing doses of Wnt3a for 2 hr, and the cytosolic fractions were analyzed by immunoblotting. ( G ) Flow cytometry analysis of cell surface FZD5/8 levels in WT or FZD5/8 DKO cells. ( H, I ) FZD5/8 double knockout abolished RSPO1-induced cytosolic β-catenin accumulation but had little effect on Wnt3a-induced increases in cytosolic β-catenin levels. The cells were treated with RSPO1 CM for 4 hr ( H ) or increasing doses of Wnt3a for 2 hr ( I ), and the cytosolic fractions were analyzed by immunoblotting. ( J ) WCLs from WT, FZD5/8 DKO and FZD5/8 DKO cells stably expressing V5-FZD5 or V5-FZD8 were analyzed by immunoblotting. ( K ) FZD5/8 double knockout abolished RSPO1-induced cytosolic β-catenin accumulation, which was restored by re-expressing V5-FZD5 or V5-FZD8. Figure 6—source data 1. Raw unedited blots for . Figure 6—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Flow Cytometry, Western Blot, Expressing, Over Expression, Stable Transfection, Double Knockout, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) U2OS cells stably expressing RNF43-HA alone or together with V5-FZD5 or V5-FZD7 were analyzed by immunoblotting with the indicated antibodies to confirm protein expression. ( B ) Cells in ( A ) were treated with increasing doses of Wnt3a for 2 hr, and the cytosolic fractions were analyzed by immunoblotting. Notably, the expression of FZD5, but not FZD7, enhanced the inhibitory effect of RNF43 on Wnt3a signaling. Figure 6—figure supplement 1—source data 1. Raw unedited blots for . Figure 6—figure supplement 1—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Stable Transfection, Expressing, Western Blot, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Verification of protein expression and biotinylation efficiency in HEK293A ZRDKO cells stably expressing V5-FZD5-mTB or V5-FZD7-mTB alone or together with RNF43ΔRING-HA. The cells were treated with or without biotin (200 μM) and analyzed by immunoblotting with the indicated antibodies. ( B ) Wnt3a or Wnt5a induces interaction between FZD5 and RNF43△RING but not FZD7. The cells in ( A ) were treated with IWP-2, control, Wnt3a or Wnt5a conditional medium with or without biotin as indicated, and the WCLs were precipitated with NeutrAvidin agarose beads, followed by immunoblotting analysis. ( C ) Schematic model of Wnt-induced FZD5/8 endocytosis and degradation. In the presence of ZNRF3/RNF43, Wnt binds to FZD5/8 and forms a complex with ZNRF3/RNF43, leading to FZD5/8 endocytosis and subsequent lysosomal degradation. This process is not affected by the absence of DVL proteins. In the absence of ZNRF3/RNF43, Wnt still induces FZD5/8 endocytosis; however, endosomes containing FZD5/8 do not merge with lysosomes, resulting in the accumulation of mature FZD5/8 intracellularly. Figure 7—source data 1. Raw unedited blots for . Figure 7—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Expressing, Stable Transfection, Western Blot, Control, Labeling

Journal: eLife

Article Title: Wnt induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

doi: 10.7554/eLife.103996

Figure Lengend Snippet: ( A ) Verification of protein expression and biotinylation efficiency in HEK293A DVLTKO cells stably expressing V5-FZD5-mTB or V5-FZD7-mTB alone or together with RNF43ΔRING-HA. The cells were treated with or without biotin (200 μM) and analyzed by immunoblotting with the indicated antibodies. ( B ) Wnt3a or Wnt5a induces interaction between FZD5 and RNF43△RING but not FZD7 in DVLTKO cells. The cells in ( A ) were treated with control, Wnt3a, or Wnt5a conditional medium with or without biotin as indicated, and the WCLs were precipitated with NeutrAvidin agarose beads, followed by immunoblotting analysis. Figure 7—figure supplement 1—source data 1. Raw unedited blots for . Figure 7—figure supplement 1—source data 2. Uncropped and labeled blots for .

Article Snippet: HEK293T, HEK293A, Huh7, MCF7, 769P, U2OS, and mouse L cells stably expressing Wnt3a or Wnt5a were cultured in high-glucose Dulbecco’s modified Eagle’s medium (boster) supplemented with 10% fetal bovine serum (Genial) and 1% penicillin/streptomycin at 37°C in a humidified incubator (Thermo Fisher Scientific) with 5% CO 2 .

Techniques: Expressing, Stable Transfection, Western Blot, Control, Labeling

Journal: Bioinformation

Article Title: Annotation of gene sequence and protein structure of brinjal EDS1

doi: 10.6026/97320630013054

Figure Lengend Snippet: Architecture of brinjal EDS1 gene and protein. SmEDS1 comprised three exons (red and yellow) and two introns (grey) with a transcription start site (TSS) at position 760. The gene codes for a 2509 base mRNA having 5’ and 3’UTR regions (yellow). Protein coding region (red) consisted of 1809 nucleotides with 42.1% GC content. The SmEDS1 protein was made up of 602 amino acids with typical features of EDS1 such as N-terminal Lipase_3 domain (protein family Pfam ID mentioned below the box) and C-terminal EP domain (evolutionary classification of protein domain structures, ECOD database ID mentioned below box).

Article Snippet: To identify the protein family, domain and functional sites the predicted SmEDS1 protein sequence was submitted to InterPro protein families database, Pfam; conserved domain database (CDD), and Evolutionary classification of protein domains (ECOD) database.

Techniques: