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. Author manuscript; available in PMC: 2016 Jul 15.
Published in final edited form as: Cell Rep. 2016 Jun 23;16(2):487–497. doi: 10.1016/j.celrep.2016.06.004

Figure 2. FOXR2 associates with MYC/MAX on chromatin through its N-terminal region.

Figure 2

(A) Upper panel: Co-IP of endogenous MAX or MYC with FOXR2 was performed with IgG or anti-FOXR2 antibody using soluble and chromatin fractions prepared from MDA-MB-468 cells. 5% of the corresponding cell lysate used in the IP was included as input control. Immunoblotting was conducted using the indicated antibodies. Lower panel: Co-IP of endogenous MAX or MYC with FOXR2 was performed with anti-FOXR2 antibody or pre-immune serum from the same mouse, using chromatin fractions prepared from MDA-MB-468 and MDA-MB-468/sh-FOXR2 cells. (B) Immunodepletion of FOXR2 and MYC performed using extracts prepared from MDA-MB-468 cells. MDA-MB-468 cell lysates were immunoprecipitated with FOXR2 or MYC antibodies three times. FOXR2, MYC, MAX, and β-actin levels were measured after each round. (C) Cell lysates of MDA-MB-468 and MDA-MB-468/sh-FOXR2 cells were immunoprecipitated with MYC or MAX antibodies and immunoblotted with the indicated antibodies. (D) HEK293T cells were transfected with constructs encoding the indicated FOX proteins. Co-IP experiments were performed using S-protein beads and blotted with antibodies recognizing the Flag-epitope tag or endogenous MYC. 5% of the corresponding cell lysate used in the IP was included as input control. Only FOXR1 and FOXR2 were able to pull down endogenous MYC. (E) Schematic diagram of MYC mutants is shown. The bHLH domain is depicted as dark grey. The mutants were: D1: (Δ1-120); D2: (Δ120-240); D3: (Δ240-350); D4: (Δ350-439). SFB-tagged wild-type and mutants of MYC were subjected to co-precipitation experiments with endogenous FOXR2 in MDA-MB-468 cells. 5% of the corresponding cell lysate used in the IP was included as input control. (F, G) Schematic diagram of FOXR2 mutants is shown. The FOX domain appears as dark grey. The mutants were: ΔN: (Δ1-113); ΔI: (Δ113-192); ΔC: (Δ192-311); D1: (Δ1-20); D2: (Δ20-40); D3: (Δ40-60); D4: (Δ60-80); D5: (Δ80-100); D6: (Δ100-113). SFB-tagged wild-type and mutants of FOXR2 were subjected to co-precipitation experiments with endogenous MYC in HEK293T cells. 5% of the corresponding cell lysate used in the IP was included as input control.