Fig 1: WLS deficiency results in ER stress and the loss of ER quality control. (A) Western blot analysis of UPR sensors, EIF2AK3-ElF2A signals, and calcium regulators in wild-type (+/+), heterozygous (fx/+), and wls-null (fx/fx) Itgax-Cre BMDCs. (B) TEM analysis of the ultrastructure and translational ribosome complex in wild-type and wls-null BMDCs. Red arrow, translational ribosomal complex. (C) intracellular calcium levels in wild-type and wls-null BMDCs following treatment with CCL2 (0.7 nM). (D-F) Western blotting analysis of WLS, ERN1, EIF2AK3, ATF6, HSPA5 CANX, and CALR in anti-WLS, anti-ERN1, and anti-EIF2AK3 immunoprecipitates probed with the respective antibodies in wild-type or wls-deficient BMDCs. Data were generated from three independent experiments. (G) Confocal immunofluorescent imaging of WLS, ERN1, EIF2AK3, and ATF6 in wild-type or wls-null BMDCs. (H-I), Western blot analysis of WLS, ERN1, EIF2AK3, ATF6, HSPA5, CANX, and CALR in anti-WLS immunoprecipitates of BMDCs treated with WNT1 and WNT3A, respectively
Fig 2: WLS-mediated molecular chaperone supercomplex modulates protein glycosylation, cytokine secretion, and cell fate in BMDCS. (A) Total glycosylation levels as measured by ELISA in BMDCs with wild-type and wls-null genotypes after TM, BFA, or DTT treatment. (B) Glycosylation levels of different kinds of glycan modification of total proteins determined by phenol-sulfuric acid method. (C) Heatmap of 507 biomarkers in wild-type and wls-null (wlsfx/fx) BMDCs; (D) Comparison of the number of hypoglycosylated proteins between wild-type and wls-null cells. #, hypo-glycosylation, no significance (*, p< 0.05); (E) GO biological process over-representation analysis with 15 signal pathways between wild-type and wls-null cells; (F) Glycosylation levels of CDH5/VE-Cadherin, BMPR1B, TGFBR1, LRP6, FZD1/Frizzled-1, FZD3, FZD4, FZD5, FZD6, and CTNNB1/ß-catenin in wild-type and wls-null cells. (G) Glycosylation levels as measured by ELISA in anti-WNT1, WNT3A, and WNT5A immunoprecipitates of wild-type and wls-null BMDC. (H) Levels of WNT1, WNT3A, and WNT5A as measured by ELISA in wild-type and wls-null BMDCs. (I) Glycosylation levels of INS (insulin), IGF1, GRB2, IGF2R, INSR, GCG (glucagon), SLC2A1/GLUT1, SLC2A2, SLC2A3, and SLC2A5 in wild-type and wls-null cells. (J-K) Levels of GCG, glucose, and ATP as measured by ELISA in wild-type and wls-null BMDCs. (L) Glycosylation levels of CD14, TLR1, TLR2, TLR3, TLR4, IFNB, IL6, and TNF in wild-type and wls-null cells. (M) Glycosylation levels of IFNA, IFNG, IFNAR1, IFNGR2, IL10, IL12A, IL23A and TGFA in wild-type and wls-null cells
Fig 3: Loss of Gpr177 blocked the extracellular secretion of WNT proteins. (a–c) Protein levels of WNT3 (a), WNT3A (b) and WNT7A (c) in germ cell culture supernatants from control, Gpr177flox/flox, Mvh-Cre; Gpr177flox/flox, Stra8-Cre and Gpr177flox/flox, Amh-Cre testes. (d–f) Secreted WNT4 (d), WNT6 (e) and WNT11 (f) examined by ELISA methods were observed in supernatants of Sertoli cells from control and three Gpr177 cKO testes. The data are expressed as the mean±S.E.M. **P<0.01
Fig 4: Effect of WLS deficiency on BMDCs. (A) Colony formation of BMDCs with wild-type, heterozygous, or wls-null (wlsfx/fx) genotypes (n = 8). (B and C) Percentage of apoptotic and autophagic BMDCs with wild-type, heterozygous, or wls-null genotypes (n = 8). (D) Western blots of autophagic markers (LC3B, PIK3C3, ATG5, ATG12 and ATG16L1) in lysates of wild-type and wls-null BMDCs (n = 3; p< 0.001). (E) Percentage of apoptotic BMDCs with wild-type and wls-null genotypes by WNT1 and WNT3A treatment. (F) Percentage of autophagic BMDCs with wild-type and wls-null genotypes after WNT1 and WNT3A treatment. (G) Level of cytokine-expressing cells in wild-type or wls-null (wlsfx/fx) BMDCs. (H) Levels of IL12A, IL6, and IL10 secreted by wild-type and wls-null BMDCs, or (I) IFNG and TNF secreted by cocultured CD4+ T cells in the presence or absence of LPS. (J) Spleen size in wild-type and DC-specific wls-null mice with or without LPS treatment. Flow cytometric analysis of the total number of (K) splenocytes and (L) DCs in wild-type and DC-specific wls-null mice after LPS treatment (n = 8; p< 0.001)
Supplier Page from CUSABIO Technology LLC for Mouse Protein Wnt-3a(WNT3A) ELISA Kit