Premade sodium dodecylsulfate polyacrylamide gel

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; Nacalai Tesque) [15 (link),16 (link),31 (link),32 (link)]. For denatured conditions, cell lysates were denatured in sample buffers (Fujifilm). The denatured samples and denatured immunoprecipitated complexes composed of primary antibody-captured antigen and Protein G resin (Thermo Fisher Scientific) were separated on premade 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 [15 (link),16 (link),31 (link),32 (link)]. The peroxidase-reactive bands were exposed on X-ray films (Fujifilm), captured using an image scanner (Canon, Tokyo, Japan), and scanned using CanoScan software (Canon). We also used a chemiluminescence scanner (C-DiGit, LI-COR, Lincoln, NE, USA) and captured immunoreactive bands through Image Studio software (LI-COR). We performed some sets of experiments in immunoblotting studies and quantified other immunoreactive bands with Image J software ver. 2.15.0.

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.