Polyvinylidene fluoride membrane

Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 3 mM MgCl₂, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) [29 (link),30 (link)]. For denatured conditions, cell lysates were denatured in sample buffers (Fujifilm). The denatured samples were separated on 10% to 15% of premade sodium dodecylsulfate-polyacrylamide gel (Nacalai Tesque or Fujifilm). The electrophoretically separated proteins were transferred to a polyvinylidene fluoride membrane (Fujifilm), blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase enzyme-conjugated secondary antibodies. The peroxidase-reactive bands were captured using CanoScan LiDE 400 (Canon, Tokyo, Japan) and scanned using CanoScan software (Ver. 1.2, Canon). We performed multiple sets of experiments in immunoblotting studies and quantified other immunoreactive bands with the control sample’s immunoreactive band as 100% with Image J software.

Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 3 mM MgCl₂, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40). For normal denatured conditions, cell lysates were denatured in sample buffers (Fujifilm), and samples were separated on a sodium dodecylsulfate polyacrylamide gel (Nacalai Tesque). The electrophoretically separated proteins were transferred to a polyvinylidene fluoride membrane (Fujifilm), blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase-enzyme-conjugated secondary antibodies. Peroxidase-reactive bands were captured using an image scanner (Canon, Tokyo, Japan) and scanned using CanoScan software (https://canon.jp/support/software/ (accessed on 1 July 2023)). The blots shown in the figures are representative of 3 blots. We performed some sets of experiments in immunoblotting studies and quantified other immunoreactive bands with one control’s immunoreactive band at 100% using the Image J software (https://imagej.nih.gov/ (accessed on 1 July 2023)).