Lide driver software

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). After centrifugation, the supernatants were mixed with sample buffer (Nacalai Tesque). Then, the samples were separated on polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred onto polyvinylidene difluoride membranes (Merck-Millipore, Darmstadt, Germany) and blocked with Blocking One, then immunoblotted with primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase. The bound antibodies were incubated with ImmunoStar Zeta (Fujifilm) and detected by X-ray film (Fujifilm) exposure. Images were captured as TIFF files using LiDE scanners (Canon, Tokyo, Japan) and processed using the accompanying LiDE driver software (Canon). The band pixels were measured in Image J software. Each image in each figure is representative of at least 3 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 sulfonyl fluoride, 1 microgram/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) [27 (link),28 (link),29 (link),30 (link)]. After centrifugation, the supernatants were incubated with a non-denaturing sample buffer (also called a native polyacrylamide gel sample buffer; Nacalai Tesque) or a denaturing sample buffer (Nacalai Tesque) for non-denaturing and denaturing conditions, respectively. Then, their 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 X-ray film (Fujifilm) exposure using an Immuno Star Zeta reagent (Fujifilm).
Images were captured as TIFF files using LiDE scanners (Canon, Tokyo, Japan) and processed using LiDE driver software (ver. 2017, Canon). The band pixels were measured using Image J software (ver. 1.53). Each image in each figure is representative of three independent experimental results (Figure S1).