SFRP2 Antibody - #DF4451

Figure 4 ERVH48-1 inhibits SFRP2 secretion to activate WNT/β-catenin signaling (A) Schematic of the strategy used to identify ERVH48-1-regulated target gene SFRP2. (B) Co-immunoprecipitation western blots for SFRP2 and ERVH48-1 or DSP-ERVH48-1 using anti-HA or anti-FLAG antibodies. 293T cells were co-transfected with vectors containing a 3xFLAG-tagged ERVH48-1 or DSP-ERVH48-1 and HA-tagged SFRP2. (C) 293T cells were co-transfected with SFRP2 (tagged with HA), with or without ERVH48-1 (tagged with 3×FLAG), or DSP-ERVH48-1 (tagged with 3×FLAG) expression plasmids as indicated for 36 h. Whole-cell extract (WCE) was used for western blot. Supernatants were collected and processed with HA-conjugated beads to pull down SFRP2-HA protein. HA antibody was used to detect exogenous SFRP2. Data were from 3 biological replicates—here, we show one of them. The right bar plot shows the quantitation of the SFRP2 protein level that was secreted into the supernatant. Data were from 3 biological replicates. Data are shown as the mean ± SEM. ∗p < 0.01, one-way ANOVA with Sidak correction between the L1 and L2, L3. (D) Schematic for the function of ERVH48-1 in sequestering SFRP2 to regulate WNT signaling. (E) Venn diagram showing proteins identified as interactors from coIP-MS for ERVH48-1 or SFRP2 interacting proteins in WT and ERVH48-1 KO cells. (F) Immunofluorescence showing the protein level of β-catenin and ERVH48-1 in WT and ERVH48-1 KO hPSCs, or in rescue lines transfected with a vector containing ERVH48-1 (R#1, R#2) or lines transfected with DSP-ERVH48-1 (DSP#1, DSP#2). Scale bars, 5 μm. (G) Western blot showing the protein level of β-catenin in WT, ERVH48-1 KO, and rescue cells transfected with wild-type ERVH48-1 (R#1 and R#2) or DSP-ERVH48-1 (DSP#1 and DSP#2). (H) The levels of β-catenin expression were quantified and compared with GAPDH. Data were from 3 biological replicates. Data are shown as the mean ± SEM. ∗∗p < 0.01, one-way ANOVA with Sidak correction between the WT and ERVH48-1 KO hPSCs, or in rescue lines transfected with a vector containing ERVH48-1 (R#1, R#2) or lines transfected with DSP-ERVH48-1 (DSP#1, DSP#2). (I) Western blot showing the protein level of β-catenin and SFRP2 in WT hPSCs cultured for 24 h with conditioned medium supernatant from WT and ERVH48-1 KO hPSCs or from rescue KO lines expressing ERVH48-1 (R#1 and R#2).