Sc 74571

Cells were grown on glass coverslips to 70 % confluence. After treatment, samples were washed twice with PBS and fixed in 4 % paraformaldehyde for 15 min. Then, cells were permeabilized with 0.1 % Triton X-100 for 10 min, followed by 10-min incubation with 5 % horse serum for blocking non-specific epitopes. AHR localization was detected with an anti-AHR antibody (Santa Cruz, sc-74571), whereas normal mouse IgG was used as control antibody. After an overnight incubation at 4 °C, samples were washed routinely with PBS and then incubated with fluorescein-conjugated secondary antibody at room temperature for 1 h. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Finally, samples were mounted and images were taken using a Zeiss fluorescence microscope (Carl Zeiss, Jena, Germany).

Whole-cell lysate in radioimmunoprecipitation assay buffer, determination of protein concentration, SDS-PAGE, and immunoblotting were performed as described previously54 (link). For immunodetection, polyvinylidene fluoride membranes were blocked with 5% non-fat milk, incubated in Tris-buffered saline (pH 7.6; Sigma). The membranes were then incubated overnight at 4 °C with specific antibodies as follows: mouse monoclonal anti-AhR (sc-74571; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-p62/SQSTM1 (sc-28359; Santa Cruz Biotechnology); rabbit monoclonal anti-LC3B (GTX127375; Genetex, Irvine, CA, USA), anti-ATG12-5 (GTX124181; Genetex), anti-ATG7 (GTX61647; Genetex), and anti-BNIP3 (ab109362; Abcam, Cambridge, UK), rabbit polyclonal anti-E-cadherin (GTX100443; Genetex) anti-vimentin (GTX100619; Genetex); and mouse monoclonal anti-β-actin (A1978; Sigma). The blots were then incubated with HRP-conjugated anti-rabbit and anti-mouse IgG antibodies obtained from Cell Signaling Technology (7074 and 7076; Danvers, MA, USA). Enhanced chemiluminescence detection was performed according to the manufacturer’s protocol (Millipore, Billerica, MA, USA).