Ecl anti rabbit igg

The antibodies used in this work were against: H3 (Abcam, ab1791, dilution 1/2000), H2B (Abcam, ab1790, dilution 1/2000), phosphorylated CHK1 (Cell Signaling, 2341 S, dilution 1/250), PCNA (Sigma, P8825, dilution 1/2500), RPA34^{62 (link)} (dilution 1/500), MCM3^{61 (link)} (dilution 1/2000), CDC45^{63 (link)} (dilution 1/1000), ELYS^{31 (link),64 (link)} (dilution 1/500), MCM4^{63 (link)} (dilution 1/1000), anti-Chk1 (dilution 1/500), anti-ORC5 (dilution 1/1000), anti-CDC6^{63 (link)} (dilution 1/500), OCT4 (Abcam, ab19857, dilution 1/500), actin (Sigma, A4700, dilution 1/500), HRP-linked ECL anti-mouse IgG (GE Healthcare, NA931V, dilution 1/4000), HRP-linked ECL anti-rabbit IgG (GE Healthcare, NA934V, dilution 1/4000) (For details see Supplementary Table 7).

Western blotting was conducted to determine the expression levels of MDM2 and CDK4 using the established cell line, with the WDLPS cell line (93T449; ATCC, Manassas, VA, USA) as a positive control and normal human foreskin fibroblast (HFF) cell line (BioWhittaker, Basel, Switzerland) as a negative control according to a previously reported procedure [30 (link)]. First, antibodies against MDM2 (1:100; Calbiochem, San Diego, CA, USA), CDK4 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (1:5000; Abcam, Cambridge, UK) were used with their corresponding secondary antibodies (ECL anti-mouse IgG and ECL anti-rabbit IgG; 1:5000; GE Healthcare). Images were obtained using an Amersham Imager 600 (GE Healthcare) after treatment with Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA). The relative expression levels of each protein were compensated for by β-actin.

The following primary antibodies were used: goat anti-ACE-2 (R&D Systems) and rabbit anti-β-actin (Proteintech Group, Tokyo, Japan). ECL™ anti-rabbit IgG (GE Healthcare, Uppsala, Sweden) or anti-goat IgG (R&D Systems) horseradish peroxidase-linked antibodies were used as the secondary antibodies. Each signal was detected using ImmunoStar Zeta or ImmunoStar LD (Fujifilm Wako) and Amersham Imager 600 series (GE Healthcare). Statistical analysis of the expression levels of each protein was performed using ImageJ Fiji (41 (link)). All actual western blotting data are in Supplementary Figure 1.

We used the following primary antibodies: first antibodies: anti-Flag (1:1000 in 5% BSA/TBST, M2, F7425, Sigma-Aldrich), anti-HA (1:2000 in 3% skim milk/TBST, clone HA-7, H9658 Sigma-Aldrich), myc (1:400 in 5% BSA/TBST, clone 9E10, sc-40, Santa Cruz Biotechnology) and GAPDH (1:1000 in 5% BSA/TBST, 14C10, rabbit mAb; Cell Signaling Technology); second antibodies: ECL anti-mouse IgG, peroxidase-linked species-specific F(ab’)2 fragment (from sheep) (1:10,000 in 3% skim milk/TBST, NA9310; GE Healthcare), ECL anti-rabbit IgG, peroxidase-linked species-specific F(ab’)2 fragment (from donkey) (1:10,000 in 3% skim milk/TBST, NA9340; GE Healthcare), and mouse TrueBlot ULTRA: anti-mouse Ig HRP (1:1000 in 3% skim milk/TBST; eBioscience). IPs used the anti-HA high-affinity (3F10; Roche) normal mouse IgG (GE Healthcare).

Right atrium samples (n = 8 patients) were homogenized in homogenization buffer and protein concentration was determined using Pierce BCA assay (Thermo Fisher Scientific). A total amount of 10 µg of protein per sample was loaded on a 4–12%—Bis-TRIS Gradient Gel (BioRad, Hercules, CA, USA) or in case of PLB a 16.5% TRIS-TRICINE-Gel (BioRad). Samples were transferred to nitrocellulose membranes (Amersham Protram) of 0.45 µm or 0.1 µm for PLB and probed with the following antibodies: GAPDH (Cell Signaling Technology #5174, Danvers MA, USA), RyR_2808_Ser (Badrilla #A010-30, Leeds, UK), phCaMKII Thr286 (Abcam #32678, Cambridge, UK), Serca2a (Badrilla #A010-20), Acetylated Lysine (Cell Signaling Technology # Ac-K-103), RyR-total (Abcam #2827), CamKII-total (Santa Cruz #sc-9035, Dallas, TX, USA), NCX (Abcam #177952), and Cav1.2 (Alomone Labs #ACC-003, Jerusalem, Israel). For secondary antibodies, HRP-conjugated ECL-Anti mouse IgG (GE Healthcare #NA931, Marlborough, MA, USA) and ECL-Anti Rabbit IgG (GE Healthcare #NA934) were used. Blots were imaged with the ChemiDoc Touch (BioRad) and band intensity was quantified using Image Lab Software 6.0.1 (BioRad).

Western blotting (WB) was performed using standard protocols. The antibodies used were: monoclonal rabbit anti-mouse phosphorylated STAT5 (p-STAT5) (Invitrogen, Thermo Fisher Scientific; 716900; dilution 1:1,000); purified mouse anti-STAT5 (BD; 610191; dilution 1:2,000); monoclonal mouse anti-mouse HSC70 (Santa Cruz Biotechnology; sc-7298; dilution 1:10,000); monoclonal mouse anti-Flag M2 ( MilliporeSigma; F3156; dilution 1:1,000); monoclonal rabbit anti–p–Aurora A (Thr288), p–Aurora B (Thr232), and p–Aurora C (Thr198) (Cell Signaling Technology; 2914; 1:1,000); monoclonal rabbit anti-Aurora B/AIM1 (Cell Signaling Technology; 3094; 1:1,000); monoclonal rabbit anti–histone H3 (anti-H3) (Cell Signaling Technology; 4499; 1:1,000); monoclonal rabbit anti–p-H3 (Ser10) (Cell Signaling Technology; 53348; 1:1,000); ECL anti-mouse IgG; HRP-linked whole antibody from sheep (GE Healthcare; NA931V; dilution 1:10,000); ECL anti-rabbit IgG; and HRP-linked whole antibody from sheep (GE Healthcare; NA934V; dilution 1:10,000). WB quantification was performed using ImageJ software (NIH). (See the complete unedited blots in the supplemental material.)