Ikkα antibody

Recombinant SAMHD1 was purified as described (42 (link)) and the pull-down assay was performed as described (14 (link)). SAMHD1 and IKKα or IKKβ were precleared with Dynabeads Protein A for 30 min and then incubated with Dynabeads Protein A and SAMHD1 antibody, IKKα antibody (61294S, Cell Signaling) or IKKβ antibody in cell lysis buffer with 0.375% CHAPS overnight at 4 °C. The beads were washed with a buffer containing 50 mM Tris-HCl PH 8.0, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate. The input and IP samples were analyzed by Western blot.

For this assay, 200 μg of total protein fraction from the prostates of treated and untreated mice was precleared with A-G beads and immunoprecipitated with agarose-conjugated IKKα antibody (Cell Signaling Technology), incubated with glutathione S-transferase-IκB substrate and subjected to SDS-PAGE as previously described [20 (link)].

Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to NC membranes, and blocked with TBST of 5% skim milk for 1 hour. The membranes were washed with TBST and then incubated with the specific primary antibody for 6 hours at 4°C. The membranes were then incubated for 1 hour at room temperature in secondary and the signal was detected by chemiluminescence (Bio rad, USA). The primary (1:1000) and secondary (1:10000) antibodies were purchased from Cell Signaling Technology, USA and listed as follows: GAPDH antibody(#2118), stat3 antibody(#12640), phospho-stat3 antibody(#98543), SAPK/JNK antibody(#9252), phospho-SAPK/JNK antibody(#4668), p65 antibody(#4764), phospho-p65 antibody(#3033), IKKα antibody(#2682), phospho-IKKα/β antibody(#2697), IκBα antibody(#4812), phospho-IκBα antibody(#2859), p38 MAPK antibody(#8690), phospho-p38 MAPK antibody(#4511), Erk1/2 antibody(#4695), phospho-Erk1/2 antibody(#4370) and HRP-linked goat anti-rabbit IgG(#7074).