rat brain protein extract  (Santa Cruz Biotechnology)

Journal: Aging Cell

Article Title: Non‐enzymatic function of WRN RECQL helicase regulates removal of topoisomerase‐I‐DNA covalent complexes and triggers NF‐κB signaling in cancer

doi: 10.1111/acel.13625

Figure Lengend Snippet: WRN‐mediated NF‐κB activation requires CHK1 and PARP1. (a) U2‐OS Cells were treated with CPT (50 nM) for indicated time periods, and activation of DDR proteins was assessed by Western blotting. (b) NEMO‐knockdown (NEMO‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of NEMO protein in NEMO‐WT (control shRNA) and NEMO‐KD cells was assessed by Western blotting. (c) NEMO‐WT and NEMO‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in terms of IκBα degradation by Western blotting. (d) ATM‐knockdown (ATM‐KD) U2‐OS cells were generated by lentivirus mediated shRNA expression system. Expression of ATM protein in ATM‐WT (control shRNA) and ATM‐KD cells was assessed by Western blotting. (e) ATM‐WT and ATM‐KD cells were treated with CPT (50 nM) for indicated time periods, and NF‐κB activation was assessed in term of IκBα degradation by Western blotting. (f) WRN‐WT cells expressing NF‐κB driven luciferase reporter were treated with CPT (50 nM) in the absence or presence of CHK1i or PARP1i for indicated time periods and NF‐κB activation was assessed in terms of fold increase in luciferase activity, which is normalized by renilla expression. All the values indicated are mean ± SD ( n = 3 for a, c, e) or mean ± SEM ( n = 4 for f). ** p < 0.01 with respect to vehicle treatment at respective time points

Article Snippet: Antibodies against p65, WRN (#SC5629), ATM (#SC23921), CHK1 (#SC8404), Lamin B (#SC6216), and CRISPR‐Cas9 double nickase plasmid (control and WRN) were from Santa Cruz biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Western Blot, Knockdown, Generated, shRNA, Expressing, Control, Luciferase, Activity Assay