Las 3000 detector

Cell lysates were incubated with the following antibodies: mouse IgG (sc-3877; Santa Cruz Biotechnology), anti-FLAG antibody (F1804; Sigma), and anti-OCT4 antibody (sc-5279; Santa Cruz Biotechnology). Immunoprecipitation was performed as described previously [39 (link)].
Western blotting was performed using the following antibodies: anti-GAPDH (sc-25778), anti-CHIP (sc-66830), anti-Nanog (sc-33759), anti-SOX2 (sc-20088), and anti-OCT4 antibodies (all purchased from Santa Cruz Biotechnology); anti-FLAG antibody (Sigma); and HRP-conjugated anti-Ub antibody (BML-PW0150; ENZO). Proteins of interest were detected using an ECL solution (Amersham Life Science) with LAS-3000 detector (Fujifilm), according to the manufacturer’s directions.

Frozen skin tissues were ground into fine powders in liquid nitrogen and homogenized after the addition of a radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) with a protease inhibitor cocktail (Calbiochem, San Diego, MA, USA). The homogenized skin tissues were sonicated and then centrifuged at 14,000× g for 20 min at 4 °C to recover proteins in the supernatant. Cells were washed twice with cold phosphate-buffered saline (PBS), lysed with the RIPA lysis buffer with 1 mM NaF, 1 mM Na₃VO₄, and a protease inhibitor cocktail on ice for 15 min, and centrifuged at 18,000× g for 15 min. SDS sample buffer (5×) was added to protein extracts from tissues, conditioned media, or cell lysates to make 1× SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). Protein samples were boiled in the presence of 100 mM β-mercaptoethanol and resolved using SDS-PAGE. Proteins in the gel were blotted onto polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The blots were then incubated with the primary and secondary antibodies. Immunoreactive signals were detected with Immobilon Western HRP substrate (Millipore) and an LAS-3000 detector (Fujifilm, Tokyo, Japan).

The cells were harvested using an SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) on ice for 15 min and centrifuged at 18,000 × g for 15 min after brief sonication. The cell lysate samples were boiled after the addition of 100 mM β-mercaptoethanol for 4 min and resolved by SDS-PAGE. Proteins in the gel were blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The blots were incubated with the indicated primary and secondary antibodies. Immunoreactive signals were detected using the Immobilon Western HRP substrate (Millipore, Billerica, MA, USA) and an LAS-3000 detector (Fujifilm, Tokyo, Japan) as previously described [66 (link)].