P53 pser15

The following antibodies were used: Lamin A/C pS22 (1:400; #2026 Cell Signaling), Cdc6 pS54 EPR759Y (1:200; ab75809 Abcam), Cyclin B1 pS126 (1:200; ab55184 Abcam, 1:100; ab3488 Abcam), p53 DO‐1 (1:500; sc‐126 Santa Cruz, Dallas, TX, USA), p53 pSer15 (1:200, #9284 Cell Signaling), p21 12D1 (1:1000; #2947 Cell Signaling), β‐tubulin 9F3 (1:1000; #2128S Cell Signaling), Cdk1 POH1 (1:1000; #9116 Cell Signaling), Cdk1 (1:200; HPA003387 Atlas antibodies), Cdk2 78B2 (1:1000; #2564 Cell Signaling), Lamin A/C (1:2000; #4777 Cell Signaling), GAPDH (1:15000‐25000; G9545 Sigma‐Aldrich), pKap1 (1:500; A300‐767A Bethyl Antibodies, Montgomery, TX, USA), and pChk2 (1:1000; #2661 Cell Signaling).

Cell were treated as for qPCR experiments. To prepare protein lysates, cells were harvested, washed, and lysed in ice cold RIPA buffer (150 mM NaCl, 5 mM Tris [pH 8.0], 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitors (Roche). Western blotting was performed according to standard protocols. Specific antibodies against AHR (NB100-2289, Novus Biologicals) MDM2 (33–7100, ThermoFisher Scientific), p21 (610233, BD Transduction), p53 (sc-126, Santa Cruz Biotechnology), p53 p-Ser33 (2526 s, Cell Signaling), p53 p-Ser15 (9286 S, Cell Signaling), p53 p-Ser37 (9289 S, Cell Signaling) were used for endogenous protein detection. As loading control, anti-β-actin (MAB1501, Millipore) or anti-GAPDH (sc-365062, Santa Cruz Biotechnology) was used after stripping the membrane with Restore Plus Stripping Buffer (ThermoFisher Scientific). Densitometric analysis of the bands was performed using ImageJ software (http://imagej.nih.gov/ij/). Full-length uncropped blots are displayed in Supplementary Fig. S4.

Protein content was measured with BCA assay (Pierce), 5–30 µg was subjected to SDS/PAGE on 4–20% acrylamide gel (Thermo Scientific) followed by transfer to nitrocellulose membrane. When detecting ATM, 7.5% Tris-HCl gel (Bio-Rad) was used, and protein was transferred to PVDF membrane (Millipore) overnight at 30 V. Membranes were blocked with 5% milk in TBS-T (Tween 0.1% wt/vol), and probed with the following antibodies: p53 pSer¹⁵ (1∶1000, Cell Signaling), p53 (1∶1000, Cell Signaling), ATM pSer¹⁹⁸¹ (1∶1000, Cell Signaling), α-tubulin (1∶2000, Santa Cruz), ERK2 (1∶2000, Santa Cruz), dCK (Clone 9D4, 1∶1000, Millipore). Polyclonal rabbit antibody against dCK pSer⁷⁴ was a gift from Dr. Francoise Bontemps (Universite Catholique de Louvain, Brussels, Belgium). ECL substrate (Millipore) was used for detection and development on GE/Amersham film. For separating nuclear and cytoplasmic lysates, NE-PER Extraction Reagents kit was used (Thermo Scientific).