Nupage novex bis tris polyacrylamide gels

Effect of miR-1 overexpression on protein level was assessed 48 hours post transfection. Preadipocytes transfected with a non-targeting control siRNA or a miR-1 mimic (20 nM final concentration) were washed with PBS and harvested in a lysis buffer consisting of 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM DTT and cOmplete Protease Inhibitor Cocktail from Roche. 30 µg protein lysates were separated by electrophoresis using 4–12% NuPAGE Novex Bis-Tris polyacrylamide gels (Life Technologies) and NuPAGE MES as running buffer. iBlot 7-Minute Blotting System form Life Technologies was used to achieve protein transfer onto nitrocellulose membranes. The membranes were blocked in 5% milk for one hour at room temperature. Incubations with primary antibodies to detect twinfilin-1 (1∶700 dilution) or α-tubulin (1∶5000 dilution) were followed by incubations with the appropriate secondary antibodies conjugated with HRP and by detection using ECL Western Blotting Detection Reagents and hyperfilms from Amersham Bioscience (Freiburg, Germany). Protein expression was quantified using ImageJ (U. S. National Institutes of Health, Bethesda, Maryland, USA) image processing software.

Cells were washed with PBS and harvested in a lysis buffer consisting of 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% TX-100, 10% glycerol, 1 mM DTT and cOmplete Protease Inhibitor Cocktail (Roche, Germany). Thirty μg protein lysates were separated by electrophoresis using 4–12% NuPAGE Novex Bis-Tris polyacrylamide gels (Life Technologies, Germany) and NuPAGE MES as running buffer. iBlot 7-Minute Blotting System from Life Technologies was used to achieve protein transfer onto nitrocellulose membranes. The membranes were blocked in 5% milk in TBST for one hour at room temperature. Incubations with primary antibodies were followed by incubations with the appropriate secondary antibodies conjugated with HRP and by detection using Amersham ECL Western Blotting Detection Reagents and Hyperfilms (GE Healthcare, Germany). Antibodies used in the study were α-tubulin (Calbiochem, Germany), β-actin, ERK 1/2 (Sigma Aldrich, Germany), AKT, IκBα, p38, pAKT (S473), pERK 1/2 (T202/Y204), pIκBα (S32/S36), pJNK (T183/Y185), pp38 (T180/Y182) (Cell Signaling, USA), JNK (BD Bioscience, USA), TRAF6 (Invitrogen, Germany), IRAK1, mouse IgG and rabbit IgG (Santa Cruz, USA).