Gtx107970

For endogenous immunoprecipitation analyses 2 mg of RS4;11, SEM cell lysates were incubated with 2 μg/mL of anti-IKK-β (GTX107970, Genetex International, USA) and IKβ-α (662402, Biolegend, USA) antibodies for 4 h at 4 °C. For recombinant immunoprecipitation analysis, 1.8 × 10⁶ HeLa cells were transiently transfected (24 h) using Lipofectamine™ 3000 (L3000015, ThermoFisher Scientific, USA) according to the manufacturer information. Plasmids used for the co-transfection are FLAG-IKK-β (NM_001556, RC219154, Origene, USA) and KCTD15 (NM_024076.2, EX-A3845-M67, TebuBio, Italy). A 2 mg of co-transfected HeLa cell extract was incubated with 2 μg/mL of anti-FLAG (F1804, Sigma Aldrich) antibody for 4 h at 4 °C. For each experiment (endogenous and recombinant immunoprecipitations) 75 µL of Protein A/G beads (sc-2003, Santa Cruz Biotechnology, Dallas, TX) in Tris–HCl 20 mM pH 7.4 + NaCl 150 mM 0.1% BSA was added and incubated for 16 h at 4 °C. Beads were washed 5 times with Tris–HCl 20 mM pH 7.4 + NaCl 150 mM and boiled for 5 min in 50 μL Laemmli buffer. Samples were loaded on SDS-PAGE and subjected to Western blot analyses. Images were acquired with the ChemiDoc Imaging System (Bio-Rad, USA) coupled with Image Lab software.

Cell extracts were prepared with M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt™; Pierce, Rockford, IL, USA) and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA) [29 (link)]. Aliquots of protein lysates were separated on SDS-8 or 15% polyacrylamide gels and transferred to PVDF membrane filters, which were blocked with 5% blotting-grade milk (Bio-Rad, Hercules, CA, USA) in TBST (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween 20) for 1 h. The filters were then incubated 1 h at room temperature with a 1:1000 dilution in TBST of antibodies against p16 (#92803, Cell Signaling Technology, Danvers, MA, USA), p21(sc-6246, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IKK2 (GTX107970, GeneTex, Hsinchu, Taiwan) and NFκB p65 (phospho Ser536) (GTX133899, GeneTex, Taiwan), reacted with corresponding secondary antibodies, and detected using a chemiluminescence assay (Millipore, Billerica, MA, USA).