Anti kap1

Cell lysates were prepared in RIPA buffer, (50 mM Tris-HCL pH 8.0, 150 mM NaCl, 1% (v/v) NP40, 0.1% (w/v) SDS and 0.5% (w/v) sodium deoxycholate, supplemented with protease inhibitors tablets and Phostop tablets) for 30 minutes on ice. Resulting lysates were clarified at 16,000 × g for 20 minutes. Gel electrophoresis was performed using the NuPAGE system (Invitrogen). Briefly, samples were resolved on 4–12% Bis-Tris gels in MOPS buffer, transferred to a PVDF membrane. Membranes were incubated with antibodies diluted in PBS supplemented with 5% milk protein and incubated, with agitation, overnight at 4° C. Membranes were washed in PBS for 3 × 5 minutes and incubated in appropriate secondary antibodies for 1 hour at room temperature. The antibodies used for immunoblotting were as follows: anti-CDK18 (Santa Cruz Biotechnology: sc-176), anti-pCDK substrate ([K/H]pSP) MultiMab rabbit Ab mix (Cell Signalling), anti-CHK1 (Sigma Aldrich), anti-CHK1 pS317 (Cell Signaling), anti-RAD9 (Bethyl laboratories: A300–800A), anti-RPA2 (Calbiochem), anti-RPA2 pT21 (Abcam), anti-RPA2 pS4/8 (Bethyl Laboratories), anti-KAP1 (Bethyl Laboratories), anti-KAP1 pS824 (Bethyl Laboratories), anti-ATR (Santa Cruz Biotechnology), anti-Actin (Abcam), HRP-secondary antibodies (DAKO) and Alexa-Fluor antibodies (Invitrogen).

Cell pellets were lysed in Laemmli buffer ×2 (Tris–HCl 0.5 M, pH 6.8; 0.5 M DTE; 0.5% SDS) and protein concentrations determined using the Bradford reagent (Bio-Rad). Protein extracts were separated on SDS–PAGE (8% or 15% or 4–15% gradient [vol/vol]) gels. Gels were transferred onto nitrocellulose membranes (GE HealthCare and Bio-Rad) for immunoblotting with the following antibodies: anti-NLRP3 (Cryo-2, 1:1,000) and anti-caspase-1 (Bally-1, 1:1,000) from AdipoGen, anti-ASC (1:2,000) from Enzo Life Science, and anti-γH2AX (JBW301, 1:1,000), anti-P-Ser15-p53 (1:1,000), and anti-ATM Ser1981 (10H11.E12, 1:1,000) from Millipore. Anti-P-KAP1 Ser824 (1:1,000), anti-KAP1 (1:1,000), and anti-NEK7 (A302-684A, 1:1,000) from Bethyl Laboratories, anti-p53 (clone DO7 1:2,000) and anti-NOXA (114C307, 1:1,000) from Santa Cruz; anti-ATM (#ab32420, 1:1,000), anti-fibrillarin (ab4566, 1:1,000), and anti-GAPDH (ab9484, 1:1,000) from Abcam; anti-FLAG (F7425 1:5,000) and anti-α-tubulin (clone B-5-1-2 1:1,000) from Sigma-Aldrich; anti-XPO2 (GTX102005 1:1,000) and anti-IPO5 (GTX114515 1:1,000) from Genetex, and anti-actin (C4, 1:100,000) from MP Biomedical. The Fiji and Image Laboratory (Bio-Rad) programs were used for densitometric analysis of immunoblots, and the quantified results were normalized as indicated in the figure legends.