Sodium dodecyl sulfate polyacrylamide gels

Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) and were prepared as described [17 (link),18 (link)]. The samples were separated on pre-made sodium dodecyl sulfate-polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany), blocked with Blocking One reagent, and immunoblotted using primary antibodies, followed by peroxidase-conjugated secondary antibodies (MBL). The bound antibodies were detected by X-ray film exposure using ImmunoStar Zeta reagent (FujiFilm). The films were captured as TIFF image files using a GT-X770 scanner (Epson, Nagano, Japan) and its driver software (Epson). The band pixels were measured in the segment analysis mode using UN-SCAN-IT software version 6 (Orem, UT, USA; URL: https://www.silkscientific.com/gel-analysis.htm). The pixel values of the protein bands were described as percentages and were compared to the control values. Images in figures are representative of experimental results.

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%.

Following the induction of differentiation for 2 days, 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 sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and 0.5% NP-40) to be collected by centrifuge as cell lysates. For denatured conditions, their supernatants were denatured sample buffer (Nacalai Tesque). The samples were separated on pre-made sodium dodecylsulfate–polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred to PVDF membranes (Fujifilm, Tokyo, Japan) sandwiched between filter papers, blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase-conjugated secondary antibodies. The Canoscan LiDE400 image scanner (Canon, Tokyo, Japan)-captured, peroxidase-reactive bands on X-ray films (Fujifilm) were analyzed using UN-SCAN-IT software (Silk Scientific, Orem, UT, USA). We performed some sets of experiments in immunoblotting studies and quantified other immunoreactive bands with the control’s immunoreactive band as 100% with NIH ImageJ software (Bethesda, MD, USA).