Enhanced ecl amersham prime western blotting detection reagent

Cells were washed twice in PBS, collected, lysed, and boiled in SDS sample buffer at 99 °C for 5 min; Equal amounts of total proteins of each cell extracts were resolved by 10–12% Bis-Tris SDS-PAGE and transferred to polyvinylidinedifluoride membranes (PVDF, Millipore, Burlington, MA, USA). Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 1–2 h. Blots were then probed with various primary antibodies, anti-Zscan4 (AB4340, Millipore, 1:1000), Dnmt1 (sc10221, Santa Cruz, 1:500), Dnmt3a (ab13888, Abcam, 1:1000), Dnmt3b (ab13604, Abcam, 1:1000), Tet2 (Kind gift from Dr. Jinsong Li from SIBS, 1:1000), H3K9me3 (ab8898, Abcam, 1:2000), H3 (ab1791, Abcam, 1:2000), and β-actin ((P30002, Abmart, 1:5000) by overnight incubation in 5% skim milk in TBST at 4 °C. Immunoreactive bands were then probed for 1–2 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary anti-Rabbit IgG-HRP (GE Healthcare, NA934V, 1:5000), or anti-mouse IgG-HRP (Santa Cruz, sc-2031, 1:5000), or anti-goat IgG-HRP (Santa Cruz, sc-2020, 1:5000). The protein bands were detected by Enhanced ECL AmershamTM prime Western blotting detection reagent (GE Healthcare, RPN2232).

Western blot was performed as described previously [20 (link)] and the antibodies used were CD133 (Biorbyt, orb10288), OCT4 (Santa Cruz, sc-9081), c-MYC (Santa Cruzs, c-47694), NANOG (Santa Cruz, sc-293121), SOX2 (Millipore, AB5603), and β-actin (Abmart, P30002). Immunoreactive bands were then probed for 2 h at room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-Rabbit IgG-HRP (GE Healthcare, NA934V), or goat anti-Mouse IgG (H + L)/HRP (ZSGB-BIO, ZB-2305). The protein bands were detected by Enhanced ECL AmershamTM prime western blotting detection reagent (GE Healthcare, RPN2232).

Western blot was performed as described previously [66 (link)] and the antibodies used were TRF1/TERF1 (Cat# ab10579, Lot# GR253870-4, RRID:AB_2201461, Abcam), OCT4/POU5F1 (Cat# sc-9081, Lot# G0607, RRID: AB_2167703, Santa Cruz), SOX2 (Cat# AB5603, Lot# 2762353, RRID: AB_2286686, Millipore), and β-actin (Cat# sc-1616R, Lot# A3009, RRID: AB_630836, Santa Cruz). The protein bands were detected by Enhanced ECL Amersham^TM prime Western blotting detection reagent (RPN2232, GE Healthcare).