Journal: bioRxiv
Article Title: Non-canonical Wnt signaling triggered by WNT2B drives adrenal aldosterone production
doi: 10.1101/2024.08.23.609423
Figure Lengend Snippet: a. WNT2B-GPC4 ectodomain, C-terminally tagged with HaloTag7 (HT7) and HPC tag, was affinity purified from conditioned media on an anti-HPC antibody matrix, and analyzed by SDS-PAGE, followed by Coomassie staining or anti-WNT2B immunoblotting (WB). Arrowhead indicates unmodified GPC4, bracket indicates glycosaminoglycan (GAG)-modified species, and asterisks indicate WNT2B protein. b. As in (a), but with WNT5A in complex with GPC4, and with anti-WNT5A immunoblotting. c. R-Spondin 3 (RSPO3, 0, 25, 100, 200 and 400ng/ml) or purified WNT5A-GPC4 complex (0.01, 0.03, 0.1, 0.3 and 1μM with respect to WNT3A) with or without RSPO3 (400ng/ml) was added to Wnt reporter cells. After 24h, Wnt pathway activity was measured by luciferase assay. Incubation with BSA served as negative control. WNT5A-GPC4 does not activate canonical Wnt signaling, even when incubated with RSPO3. Points represent average activation for two biological replicates, normalized to untreated cells, and error bars represent SD. d. SFRP2 (1μM) was added in serum-free media in WNT3A- or WNT2B-expressing HEK293 cells. Serial dilutions of the conditioned media were then added to Wnt reporter cells, and Wnt pathway activity was measured by Dual-Glo luciferase 24h later. BSA (1μM) served as negative control. WNT2B released by SFRP2 is unable to activate canonical Wnt signaling, in contrast to WNT3A-SFRP2 conditioned media. Points represent average activation for two biological replicates, normalized to the negative control, and error bars represent SD. e. As in (d), but WNT-expressing cells were incubated with 1μM of GPC4. f. As in , but purified WNT3A-GPC4 complex (1μM) was mixed with the indicated concentrations of GPC4 alone or in complex with WNT3A, WNT5A or WNT2B. WNT3A-SFRP2 activity is abolished by WNT5A-GPC4 and WNT2B-GPC4 complexes in a dose-dependent manner, which contrasts GPC4 alone or WNT3A-GPC4 complex. g. Extracellular domains (ECD) of ROR1 and ROR2, N-terminally tagged with a FLAG tag, were affinity purified from conditioned media on an anti-FLAG antibody matrix. Purified proteins were analyzed by SDS-PAGE and Coomassie staining. h. As in (a), but with WNT2B in complex with SFRP2, C-terminally tagged with 8x-His tag and HPC taI. i. As in (b), but with WNT5A in complex with SFRP2. j. Purified SFRP2 (5μM) was incubated with FLAG-tagged ROR1-ECD (2.5μM), followed by immunoprecipitation with antibodies against the FLAG tag. Samples were analyzed by SDS-PAGE and immunoblotting. SFRP2 does not interact with ROR1-EcD. k. Activity of RhoA in cell lysates of HEK293 cells treated for 6h with GPC4 alone or in complex with WNT3A, WNT5A or WNT2B (2μM) was assessed by Rhotekin-RBD pull-down assay. RhoA endogenous levels are shown in the lysates. Both WNT5A-GPC4 and WNT2B-GPC4 complexes induce activity of RhoA, in contrast to GPC4 alone or in complex with WNT3A. Blotting for α-tubulin served as loading control. l. HEK293 cells were co-transfected with the firefly luciferase reporter (pGL4.34) and the renilla luciferase thymidine kinase reporter (pRL-TK). They were then used to assay RhoA activation by purified GPC4 alone or in complex with WNT3A, WNT5A, and WNT2B (1 μM). We found that the activity of RhoA is induced by WNT2B-GPC4 or WNT5A-GPC complexes, but not by WNT3A-GPC4 or GPC4 alone. The bars represent the average from three independent experiments performed in duplicate, normalized to untreated cells. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (ns, not significant; **p < 0.01; ***p < 0.001). Data are represented as mean ± SEM.
Article Snippet: After washing the beads three times with 2mM CaCl and 0.2% DDM, bound proteins were eluted in elution buffer (20mM HEPES, pH 7.5; 200mM NaCl; 5mM EDTA; 100μg/mL FLAG or HPC peptide) and were analyzed by SDS-PAGE followed by immunoblotting using rabbit monoclonal or polyclonal antibodies against WNT3A (Cell Signaling, #2721S), WNT5A/B (Cell Signaling, #2530S) or, WNT2B (Abcam, #ab203225), and anti-mouse monoclonals against FLAG-M1 and anti-HPC, a generous gift from Andrew C Kruse (Harvard Medical School).
Techniques: Affinity Purification, SDS Page, Staining, Western Blot, Modification, Purification, Activity Assay, Luciferase, Incubation, Negative Control, Activation Assay, Expressing, FLAG-tag, Immunoprecipitation, Pull Down Assay, Control, Transfection
