Enhanced chemi luminescence (ecl)

Whole-cell extracts were lysed in pre-cold lysis buffer with freshly added protease/phosphatase inhibitors. After 30 mins, the lysates were then spun at 13,300 rpm for 10 minutes and the pellet was discarded. Equal amount of protein were loaded for electrophoresis and then electro-transferred to a nitrocellulose membrane (Biorad, USA), which was blocked with Blocking One (Nacalai Tesque, Inc., Japan), and probed with the primary antibodies of interest overnight at 4°C. The blot was washed, exposed to HRP-conjugated secondary antibodies (Santa Cruz Co, CA) for 1 h, and finally examined by chemiluminescence (ECL, advansta Inc., USA).

Collected tissues were homogenized in cell lysis buffer (Beyotime Institute of Biotechnology), with protease inhibitors (Biotool).The homogenates were centrifugated for 10 min (12,000 g), and protein concentration in the supernatant was measured using the BCA Protein Assay Kit (BIOMIGA). Equal amounts of total protein (40–60 μg) were loaded and separated on 12% SDS-PAGE by electrophoresis (120 V, 90–120 min). Proteins on SDS-PAGE were blotted onto PVDF membranes (100 V, 90–120 min). The membranes were blocked with 5% skim milk dissolved in TBST (20mMTris-HCl,150mMNaCl,0.05%Tween-20) solution for 2 h, and incubated sequentially with primary antibodies in TBST against IL-6 (1:10000, abcamab7737), IL-1β (1:2000, R&D Systems, AF-401-NA), TNF-α (1:1000, abcamab1793), Wnt5a (1:2500, abcamab72583), α-tubulin (1:10000, Proteintech,66031-1-lg) or GAPDH (1:10000, Santa Cruz sc-25778) for 2 h.After washes with TBST3 times for 10 minutes each and the membranes were incubated with goat anti-rabbit IgG (1:30000, abcamab97051) or goat anti-mouse IgG (1:30000 abcamab97023) secondary antibody conjugated with horseradish peroxidase (HRP) in TBST, After washes with TBST3 times for 10 minutes each and the protein bands were visualized by ECL (Advansta). Tubulin and GAPDH were used as loading controls.

Proteins from tissues and cultured cells were extracted by the lysis buffer [20 mM Tris/HCl pH 7.4, 50 mM NaCl, 10% (w/v) Glycerol, 0.1 mM EDTA, 2% SDS] supplemented with cOmplete protease inhibitor (Roche) and 1 mM PMSF. The protein concentration was quantified using Pierce 660 nm Protein Assay Reagent (Thermo Scientific, Cat. 23,225). Proteins were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore), and detected with primary and secondary antibodies listed in Additional file 1: Table S2 using ECL (Advansta, Cat. K-12,045-D50) and ChampChemi910 (Beijing Sage Creation Science Co., Ltd.).

Cell lysates were separated by SDS-PAGE, and the proteins were then transferred to PVDF membranes. The proteins were detected using antibodies against the following: RB (ab181616, 1:2000), cyclin D1 (ab40754, 1:1000), E2F1 (ab179445, 1:1000) (Abcam, Cambridge, UK), phospho-RB (p-RB) (S780) (#8180, 1:1000), PARP/cleaved PARP (#9542, 1:1000) (Cell Signaling Technology), and β-Actin (AC026, 1:100,000) (ABclonal, Boston, USA). Specific bands were visualized by ECL (Advansta, USA) and detected with an imaging system (Bio-Rad, USA).

WB was performed as described previously^{4 (link)}. In brief, cells grown in six-well tissue culture plates in medium supplemented with 10% CD-FBS to reduce the endogenous E2 concentration for 48 h were transfected with an siRNA specific for CXXC5 or a non-target control siRNA (AllStar, CtS) in the absence (EtOH, 0.01%) or the presence of E2 (10⁻⁸ M) for 48 h. At the termination, cells were collected and the nuclear content was isolated using NE-PER protein extraction kit (Thermo Fisher). Protein concentration was measured with Bradford Protein Assay (Bio-Rad). Nuclear extracts (50 μg) were then subjected to 12% SDS-PAGE. Proteins were probed with an antibody specific to CXXC5 (ab106533, Abcam) or PARP1 (9542; Cell Signaling Tech.) followed by a secondary antibody conjugated with horseradish peroxidase (Advansta Inc., San Jose, CA, USA). The HDAC1 antibody (Abcam, ab19845) was used for monitoring the levels of HDAC1, which was used as the loading control. Proteins were visualized with ECL (Advansta) and images were captured with ChemiDoc Imaging System (Bio-Rad). PageRuler Prestained Protein Ladder (Thermo-Fisher) was used as a molecular weight marker.