Mini protean electrophoresis chamber

After cell lysis in 1% (v/v) NP-40, soluble fractions were analyzed by western blot under non-reducing conditions, using a Biorad Mini-Protean electrophoresis chamber for SDS-PAGE and transfer on polyvinylidene fluoride (PVDF) membranes. Membranes were probed with anti-mouse CD81 MT81^{11 (link)} at 2 µg/ml, anti-mSR-B1 polyclonal antibody (Ab24603) diluted at 0.9 µg/ml, and anti-mouse GADPH (TAB1001) as a loading control (0.5 µg/ml). Chemiluminescence detection was performed using ECL Prime reagents (RPN2232,GE healthcare Life sciences) and an ImageQuant LAS 4,000 system (GE Healthcare).

White cells from bone marrow of control and Hdac7^fl/− mice were extracted as in sorting procedure. Next, cells were stained with anti-CD19-microBeads (Miltenyi Biotec) according to manufacturer's instructions. CD19⁺ B cells were isolated by magnetic separation with LS column adapters (Miltenyi Biotec). Purified cells were lysed with RIPA buffer. Lysates were resolved on 8–15% SDS-PAGE (Mini-Protean electrophoresis chamber, Bio-Rad) and transferred on nitrocellulose membranes (Amersham Biosciecnes). Membranes were blocked in 5% milk in TBS with 0.1% Tween (TBS-T) and incubated overnight at 4°C, with primary antibodies (anti-Tet2 ab94580, abcam 1:1000; anti-HDAC7 sc-11421, Santa Cruz Biotechnology 1:1000; anti-H3K9me3 ab8898 (Abcam) 1:1000; anti-PUMA 12450 (Cell Signaling) 1:500, anti-IRF4 sc-48338 (Santa Cruz Biotechnology) 1:500; anti-c-MYB sc-74512 (Santa Cruz Biotechnology) 1:500; anti-H3 ab1791 (Abcam) 1:1000; anti-Lamin B1 ab16048 (Abcam) 1:1000, and anti-Actin AC-15 (Sigma-Aldrich) 1:40000). Secondary antibody incubations (HRP-anti mouse, P0260, or anti rabbit, P0448, Dako 1:3000), were carried out for 1h at room temperature. Protein signal was detected using ECL western detection kit (Amersham Biosciences).

Western blots were performed as before (29 (link),30 (link)). Briefly, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 250 mM NaCl, 2% Nonidet-P40, 2.5 mM EDTA, 0.1% SDS, 0.5% DOC) supplemented with Halt PIC (Thermo, 78430) and PMSF (Sigma, 93482), lysates were quantified by BSA assay and resolved on 7–9% SDS-PAGE gel (Mini-PROTEAN electrophoresis chamber, Bio-Rad), and transferred to PVDF membranes (Mini Trans-Blot apparatus, Bio-Rad) in 5-10% methanol transfer buffer following manufacturer's protocols. Membranes were blocked in 5% milk in PBS with 0.1% Tween-20 (PBS-T) and incubated O/N at 4°C with primary antibodies (anti-Tet1 1:3000 (GeneTex, GTX125888), anti-Tet2 1:750 (Abcam, ab124297), anti-Lamin-b1 1:1000 (ABclonal, A1910), anti-Sin3a 1:1000 (Abcam, ab3479), anti-Ezh2 1:1000 (CST, 5246), anti-Chd4 1:1000 (Abcam, ab70469)). Secondary antibody incubations (HRP-anti-mouse, 401253, or HRP-anti-rabbit, 401393, Calbiochem 1:2500) were for 1 h at RT. Protein bands were identified with ECL chemiluminescence reagent (Amersham RPN2106). Lamin-b1 was used as loading control.

The cells were lysed in a Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 2% Nonidet-P40, 2.5 mM EDTA, 0.1% SDS, 0.5% DOC Sigma-Aldrich, Inc., St. Louis, MO, USA) supplemented with PIC and PMSF. The lysates were resolved on an 8–12% SDS-PAGE (Mini-PROTEAN electrophoresis chamber, Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Mini Trans-Blot apparatus, Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocols. The membranes were blocked in 5% milk in PBS with 0.1% Tween-20 (PBST) and incubated overnight at 4 °C or for 1 h at room temperature with primary antibodies (p21: BD Pharmingen 556431; p53: CST2524T; p27: Santa Cruz sc1641; CycD1: CST2978T; Cdk1: Abcam ab18; Cdk2: CST2546T; β-actin: Abcam ab6276). Secondary antibody incubations (HRP-anti-mouse Calbiochem 401253 or HRP-anti-rabbit Calbiochem 401393, 1:3000) were carried out for 1 h at room temperature. In all experiments, β-actin was used as the loading control. The quantification of the Western blot band signal intensities was performed using Image J (v.1.53k) and the data were normalized to the respective actin signal intensities and then plotted.

Heart and cell homogenates (30 μg protein), or isolated cytosolic (20 μg protein), mitochondrial (15 μg protein), and nuclear (20 μg protein) fractions were electrophoresed on a 4–15% Mini-Protein TGX gel using a Mini-PROTEAN electrophoresis chamber (Bio-Rad), and proteins were transferred to a PVDF membrane using a Criterion Trans-blot System (Bio-Rad). Membranes were blocked with 5% non-fat dry milk (NFDM) in 50 mM Tris-HCl (pH 7.5) / 150 mM NaCl (TBS), washed with TBS-0.1% Tween 20 (TBST) and TBS, and incubated overnight at 4°C with antibody clones listed in Table C of S1 Table. Antibodies from Santa Cruz were used at 1:200 dilutions, and all other antibodies were used at 1:1000 dilution in TBST-5% NFDM. Membranes were washed with TBST and TBS, incubated with HRP-conjugated secondary antibody (1: 10,000 dilution, Southern Biotech, Birmingham AL), and images were acquired by using an Image Quant LAS4000 system (GE Healthcare, Pittsburgh MA). Immunoblots were subjected to Ponceau S staining to confirm equal loading and transferring of samples. Densitometry analysis of protein bands was performed using a Fluorchem HD2 Imaging System (Alpha Innotech, San Jose CA), and normalized against GAPDH (tissue homogenates and cytosolic fractions), COIV (mitochondrial fractions) or Lamin B (nuclear fractions).