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. 2015 Sep 3;97(3):445–456. doi: 10.1016/j.ajhg.2015.08.002

Figure 3.

Figure 3

ARID1B Associates with β-Catenin in Nuclear Puncta and Represses Wnt/β-Catenin-Dependent Transcription through BRG1

(A) Representative immunofluorescence pictures of U2OS cells expressing transfected ARID1B and GFP β-catenin, alone (rows 1 and 2) or together (row3), stained with an antibody against ARID1B. Note that the antibody strongly recognizes overexpressed ARID1B as compared to endogenous ARID1B in these cells.

(B) Western blotting (WB) for indicated endogenous proteins of lysates (INPUT) and IgG (control) or β-catenin immunoprecipitates (IP) from HCT116 nuclei. Enrichment in nuclear proteins was confirmed by the presence of histone H3 (H3) and absence of α-tubulin. Asterisk indicates heavy chains IgG, showing equal loading of IP reactions. Molecular weights (kDa) are indicated on the left of the panels.

(C) Representative immunofluorescence pictures of proximity ligation assays (PLAs) for endogenous ARID1B and β-catenin in HCT116 cells transfected with indicated siRNAs.

(D) Quantification from experiments conducted in (C).

(E) Representative immunofluorescence pictures of U2OS cells co-expressing transfected ARID1B and GFP β-catenin in the absence or presence of co-transfected Flag BRG1 (+ Flag BRG1) stained with an antibody against ARID1B.

(F) Immunofluorescence pictures of PLAs for endogenous ARID1B and β-catenin in HCT116 cells transfected with indicated siRNAs.

(G) Quantification from experiments conducted in (F).

(H) Ratio of TOP/FOPFlash in SW480 cells transfected with indicated plasmids in the presence of co-transfected siRNAs targeting GFP (siControl) or BRG1 (siBRG1). In panels (A) and (E), right columns show merged pictures with DAPI and inlets show magnified nuclear regions. In panels (D) and (G), n shows the number of nuclei analyzed, error bars show SEM, and double asterisks denote p <0.01 for differences from controls (siGFP), with unpaired Student’s t test, from three independent experiments. In panel (H), error bars show SEM and single asterisks denote p< 0.05 for differences from controls (Flag + siControl) from at least three independent experiments.