Hrp conjugated anti β actin antibody

Tumor tissues were homogenized using a BeadBug Homogenizer (Benchmark Scientific, Sayreville, NJ). Protein lysates were prepared with lysis buffer (1% Triton X-100, 0.05% SDS, 100 mM Na₂HPO4, and 150 mM NaCl). Protein lysate was electrophoresed on a 4–20% pre-cast SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred onto Amersham Hybond 0.45 PVDF membranes (GE Healthcare, Chicago, IL). After blocking with 5% non-fat milk in PBS-0.05% Tween 20, the membranes were incubated with primary antibodies at 4°C overnight, and then secondary antibodies for 1 h at room temperature. Antibodies include anti-p-ATR antibody (#2853, Cell Signaling Technology), anti-ATR antibody (#13934, Cell Signaling Technology), p-CHK1 antibody (#2348, Cell Signaling Technology, Danvers, MA), cleaved-caspase-3 antibody (#9664, Cell Signaling Technology), HRP-conjugated anti-β-actin antibody (#HRP-6008, ProteinTech), anti-rabbit secondary antibody (#7074, Cell Signaling Technology). The blots were developed using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad).

Cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Seradigm, Radnor, PA) and penicillin/streptomycin (Life Technologies) at 37°C and 5% CO₂. They were treated with a medium containing elimusertib (0.3 μM or 3 μM) or DMSO solvent (0.03%) which functioned as the untreated control. At the desired time points, cells were trypsinized and collected for preparing protein lysates. Protein lysates were prepared with lysis buffer (1% Triton X-100, 0.05% SDS, 100 mM Na₂HPO4, and 150 mM NaCl) and then electrophoresed on a 4-20% pre-cast SDS-polyacrylamide gel (Bio-Rad) and transferred onto Amersham Hybond 0.45 PVDF membranes (GE Healthcare, Chicago, IL). After blocking with 5% non-fat milk in PBS-0.05% Tween 20, the membranes were incubated with primary antibodies at 4°C overnight, and then secondary antibodies for 1 h at room temperature. Antibodies include anti-p-ATR antibody (#2853, Cell Signaling Technology), anti-ATR antibody (#13934, Cell Signaling Technology), p-CHK1 antibody (#2348, Cell Signaling Technology), cleaved-caspase-3 antibody (#9664, Cell Signaling Technology), HRP-conjugated anti-β-actin antibody (#HRP-6008, ProteinTech), anti-rabbit secondary antibody (#7074, Cell Signaling Technology). The blots were developed using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad).

Tumor tissues were homogenized using a BeadBug Homogenizer (Benchmark Scientific). Protein lysates were prepared with lysis buffer (1% Triton X-100, 0.05% SDS, 100 mM Na₂HPO4, and 150 mM NaCl). Protein lysate was electrophoresed on a 4–20% pre-cast SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred onto Amersham Hybond 0.45 PVDF membranes (GE Healthcare, Chicago, IL). After blocking with 5% non-fat milk in PBS-0.05% Tween 20, the membranes were incubated with primary antibodies at 4°C overnight, and then secondary antibodies for 1h at room temperature. Antibodies include anti-p-MAP2K4 antibody (#9156, Cell Signaling Technology), anti-MAP2K4 antibody (#9152, Cell Signaling Technology), anti-p-JNK antibody (#9255, Cell Signaling Technology), anti-JNK antibody (#9252, Cell Signaling Technology), anti-p-p38 antibody (#9211, Cell Signaling Technology), anti-p38 antibody (#8690, Cell Signaling Technology), anti-PARP-1 antibody (#9532, Cell Signaling Technology), HRP-conjugated anti-β-actin antibody (#HRP-6008, ProteinTech), cleaved-caspase-3 antibody (#9664, Cell Signaling Technology), anti-rabbit secondary antibody (#7074, Cell Signaling Technology). The blots were developed using Clarity or Clarity Max Western ECL Blotting Substrates (Bio-Rad).