Denaturing sample buffer

Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl₂, 1 mM dithiothreitol, 1 mM phenylmethane sulfonyl fluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) [11 (link),12 (link)]. For denaturing conditions, their supernatants were denatured in a denaturing sample buffer (Nacalai Tesque). The samples were separated into 8 to 12% sodium dodecyl sulfate–polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred to PVDF membranes (Fujifilm), blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase-conjugated secondary antibodies (MBL, Tokyo, Japan). A CanoScan LiDE400 image scanner (Canon, Tokyo, Japan) captured the peroxidase-reactive bands on X-ray films (Fujifilm). Quantified immunoreactive bands were compared with the control’s immunoreactive bands, which were set to 100%, using Image J software (NIH, Bethesda, MD, USA).

Cells were lysed in lysis buffer A (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl₂, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) [15 (link),16 (link)]. For non-denatured and denatured conditions, the supernatants were incubated with non-denaturing sample buffer (also called native sample buffer; Nacalai Tesque, Kyoto, Japan) and denaturing sample buffer (Nacalai Tesque, Kyoto, Japan), respectively, after centrifugation [13 (link),14 (link),15 (link)]. The samples were then separated on non-denatured or denatured polyacrylamide gels (also called pre-made PAGE gels; Nacalai Tesque, Kyoto, Japan). The electrophoretically separated proteins were transferred onto polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany) and blocked with Blocking One reagent, then immunoblotted with primary antibodies followed by secondary antibodies conjugated with HRP proteins. The bound antibodies were detected by means of X-ray film exposure using ImmunoStar Zeta reagent (Fujifilm)
Images were captured as TIFF files using a Canon LiDE 400 scanner (Canon, Tokyo, Japan) and processed using the accompanying driver software (Canon, Kyoto, Japan). The band pixels were measured in Image J software. Each image in each figure is representative of three independent experimental results.

Cells were lysed in lysis buffer A (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl₂, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) [23 (link),24 (link)]. After centrifugation, for non-denatured and denatured conditions, the supernatants were incubated with a non-denaturing sample buffer (also called the native polyacrylamide gel sample buffer; Nacalai Tesque) and the denaturing sample buffer (Nacalai Tesque), respectively. Then, the samples were separated on non-denaturing or denaturing polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred onto polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany) and blocked with Blocking One reagent, then immunoblotted with primary antibodies followed by secondary antibodies conjugated with HRP proteins. The bound antibodies were detected by means of X-ray film exposure using ImmunoStar Zeta reagent (Fujifilm). Images were captured as TIFF files using Canon LiDE scanners (Canon, Tokyo, Japan) and processed using the accompanying driver software (Canon). The band pixels were measured in Image J software. Each image in each figure is representative of three independent experimental results.