Anti pa16 tag nz 1

Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). The proteins were subjected to electrophoresis on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), each membrane was incubated with primary mAbs, such as 1 μg/mL of PMab-219, 1 μg/mL of anti-PA16 tag (NZ-1), or 1 μg/mL of anti-β-actin (AC-15; Sigma-Aldrich Corp., St. Louis, MO, USA), and subsequently with peroxidase-conjugated anti-mouse IgG (1:1000; Agilent Technologies, Santa Clara, CA, USA) or anti-rat IgG (1:10000; Sigma-Aldrich Corp.). Bands were visualized with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Cell lysates (10 μg) were boiled in a sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). The proteins were electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), each membrane was incubated with primary mAbs, including 1 μg/mL of PMab-233, 1 μg/mL of anti-PA16 tag (NZ-1), or 1 μg/mL of anti-β-actin (AC-15; Sigma-Aldrich Corp., St. Louis, MO, USA), and subsequently with peroxidase-conjugated anti-mouse IgG (1:1000; Agilent Technologies, Santa Clara, CA, USA) or anti-rat IgG (1:10000; Sigma-Aldrich Corp.). The developed bands were visualized with the ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).