Canoscan 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; 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.

Collected cells were lysed in lysis buffer [36 (link),37 (link),38 (link)]. Their centrifugally collected supernatants (20 mg per sample) were denatured in sample buffers (Fujifilm). They were separated on a sodium dodecylsulfate (SDS) polyacrylamide gel. The cell lysate-derived proteins or the resultant precipitates were separated using polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Fujifilm). They were blocked with Blocking One (Nacalai Tesque) and immunoblotted using the respective primary antibodies and peroxidase enzyme–conjugated secondary antibodies.
Enzymatically reactive bands were detected using CanoScan LIDE400 (Canon, Tokyo, Japan) and scanned using CanoScan software (ver. 2023, Canon). The blots shown in the figures are representative of 3 blots. We performed multiple sets of experiments and quantified other bands with one control’s band as 100% using Image J software (ver. Java 8, https://imagej.nih.gov/ accessed on 5 December 2023).

Cells were lysed in lysis buffer [13 (link),14 (link),15 (link),16 (link)]. Under normal denaturing conditions, cell lysates were denatured with sample buffer (Fujifilm), and samples were separated on sodium dodecyl sulfate-polyacrylamide gels (Nacalai Tesque). Separated proteins were transferred to polyvinylidene fluoride membranes (Fujifilm), blocked with Blocking One solution (Nacalai Tesque), and immunoblotted using a primary antibody followed by a peroxidase enzyme-conjugated secondary antibody. Peroxidase-reactive bands were captured using a CanoScan image scanner (Canon, Tokyo, Japan) and scanned using CanoScan software (Canon). The blots shown in the figure are representative of three blots. Several sets of experiments were performed in immunoblot studies, and Image J software (https://imagej.nih.gov/, accessed on 1 May 2023) was used to quantify immunoreactive bands with one control immunoreactive band as 100%.