Goat anti rabbit hrp antibodies

Cell fractionation was performed using the nuclear extract kit according to the manufacturer's instruction (Active Motif, Carisbad, CA, USA). Protein concentrations were determined using the Pierce BCA assay (Thermo Scientific, Reinach, Switzerland); 5 μg were loaded per lane on a 4–20% Mini-PROTEAN TGX Gel (Bio-Rad Laboratories AG, Reinach, Switzerland). Separated proteins were transferred to PVDF membranes using the transfer turbo blot system (Bio-Rad). Monoclonal antibodies used in this study were directed against TNFAIP3 (clone 59A426, diluted 1 : 100, Abcam, Cambridge, UK), NKIRAS2 (clone ab57303, diluted 1 : 500, Abcam), IκBα (clone E130, diluted 1 : 10 000, Abcam), phospho-IκBα (clone 5A5, diluted 1 : 2000, Cell Signaling, Beverly, MA, USA), α-tubulin (clone B512, diluted 1 : 2000, Sigma-Aldrich), histone 3 H3 (clone D1H2, diluted 1 : 2000, Cell Signaling) and p65 (polyclonal, diluted 1 : 2000, Abcam). Secondary goat anti-mouse-HRP and goat anti-rabbit HRP antibodies (Bio-Rad) were used at 1 : 5000 or 1 : 7000, respectively. Protein levels from total or cytoplasmic extracts were normalized to α-tubulin and protein levels from nuclear extracts were normalized to histone 3. Quantification of protein bands were performed using a luminescent image analyzer LAS-4000 (Fujifilm, Dielsdorf, Switzerland) and Multi Gauge software (Fujifilm Version 3.0).