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. 2021 Dec 31;109(1):97–115. doi: 10.1016/j.ajhg.2021.11.018

Figure 7.

Figure 7

WNT5B inhibits E2-induced ALPL expression via SIN3A

(A and B) mOBs (A) or U2OS-ERα cells (B) were treated with EtOH or 10 nM E2 in the presence or absence of 50 ng/mL rWNT5B for 4 days. Then, alkaline phosphatase (ALP) activity was detected and was normalized with total protein.

(C) Expression of ALPL also was measured in U2OS-ERα cells.

(D) Diagram represents ALPL enhancers in U2OS-ERα cells. U2OS-ERα cells were treated with PBS/EtOH (Ctrl), 10 nM E2, 50 ng/mL rWNT5B, or E2 with rWNT5B for 45 min.

(E and F) ChIP was performed using an antibody to (E) ERα or (F) SIN3A. qPCR was performed with primers to the ALPL enhancers and normalized to the β-actin (ACTB) promoter.

(G) Co-immunoprecipitations were performed with an antibody to SIN3A before blotting with an antibody to ERα or IgG in U2OS-ERα cells.

(H) mOBs were treated with 50 ng/mL rWNT5B for 0 and 15 min, and then were incubated with or without calf intestinal protease (CIP) in vitro before detecting phospho-CDK5 (Tyr15) and SIN3A by immunoblotting and normalized to β-actin (BA) by ImageJ. Three different biological replicates were tested and a representative image from one replicate is presented. n = 3, Student’s t test: ∗∗p < 0.01, ∗p < 0.05.