Ab152146

The proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, P.R. China) and phenylmethanesulfonyl fluoride (PMSF; Beyotime) according to the manufacturer’s protocol. The concentration of proteins was then estimated by BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Equivalent proteins were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Thereafter, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% skim milk (Nestlé, Shuangcheng, P.R. China). After rinsing, the membranes were incubated at 4°C overnight with primary antibodies against FZD8 (ab155093), cyclin D1 (ab137875), c-Myc (ab152146), β-catenin (ab6302), T-cell transcription factor 4 (TCF-4; ab185736), or GAPDH (ab128915) (all from Abcam, Cambridge, MA, USA). After rinsing again, the membranes were incubated with secondary antibody marked by horseradish peroxidase for 1 h at room temperature. The membranes were washed again and then transferred into the Bio-Rad ChemiDoc™ XRS system, followed by adding 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore) to cover the membrane surface. The signals were captured using Image Lab™ Software (Bio-Rad, Hercules, CA, USA).

Bacteria were resuspended in 800 μL co-IP lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5% glycerol, pH 7.4) and 120 μg lysozyme was added. The samples were incubated at 37 °C under shaking at 1400 rpm for 1 h. Afterwards, 8 μL 10% NP-40 solution was added and the bacteria were lysed by ultrasonication (5 × 30 s, 80%, on ice during breaks). The insoluble fraction was pelletized (10 000g, 30 min, 4 °C) and the supernatant was sterile filtered through a 0.2 μm PTFE filter. Protein concentration was determined using a BCA assay (Roti-Quant universal, Carl Roth GmbH + Co. KG). 30 μL Protein A/G agarose beads (Thermo Fisher Scientific) were transferred to protein low-bind microcentrifuge tubes (Eppendorf) and washed with 1 mL co-IP wash buffer (50 mM Tris–HCl, 150 mM NaCl, 5% glycerol, 0.05% NP-40, pH 7.4) and centrifuged for 1 min at 1000g at 4 °C. 500 μg proteome (in 500 μL) and either 1 μL anti-c-Myc antibody (rabbit polyclonal, ab152146, 1 mg mL⁻¹, Abcam) or 0.4 μL rabbit mAb IgG isotype control (2.5 mg mL⁻¹, Cell Signaling Technology, Danvers, United States) were added. The samples were incubated at 4 °C for 3 h under constant rotation. The supernatant was removed after centrifugation (1000g, 1 min, 4 °C), and the beads were washed twice with 1 mL co-IP wash buffer. The detergent was removed by washing the beads twice with co-IP lysis buffer.