Anti ha tag

At sacrifice, mouse heart samples were frozen by immersion in isopentane cooled in liquid nitrogen. For immunohistochemistry analysis, heart samples were mounted in optimal cutting temperature compound (OCT) and sectioned on a cryostat at −20 °C to a thickness of 8 μm. Sections were fixed in 10% neutral buffered formalin (SIGMA, HT501128) at RT. After three washes in PBS, samples were permeabilized using 0.2% Triton X- 100 in PBS for 15 min at RT, followed by three washes in PBS, and then blocked in 5% normal goat serum + 2% BSA in PBS for 1 h at RT. After three washes in PBS, samples were incubated with the rabbit anti-HA-Tag (Cell Signaling, 3724) primary antibody 1:2000 in DAKO solution (Agilent, S3022) for 1 h at RT, followed by three additional washes in PBS. Sections were then incubated with goat anti-rabbit IgG (H + L) AlexaFluor diluted 1:300 in DAKO diluent solution 568 for 1 h at RT, washes three times in PBS and finally coverslip with prolonged diamond antifade mountant with DAPI (Thermo Fisher, P36962). All confocal images were acquired using a Zeiss LSM880 microscope using identical acquisition parameters.

After 48 hrs of doxycycline induction, 10 μg total protein was boiled for 5 mins in Laemmli buffer (125 mM Tris pH6.8, 5% β-Mercaptoethanol, 2% SDS, 10% Glycerol, 0.002% Bromophenol Blue). Samples were loaded per lane onto SDS-PAGE gels alongside 3 μl BluEye Prestained protein ladder (GeneFlow). Gels were transferred to PVDF membrane (GE Healthcare) at 350 mA for 1 hr. Membranes were blocked in TBS-T buffer (1x TBS (Severn Biotech), 0.1% v/v Tween-20 (Sigma)) containing 5% non-fat milk powder for 1 hr at room temperature. Primary antibodies were incubated in TBS-T with 1% milk as follows; M2 anti-FLAG (Sigma), anti-CASP3 and anti-HA tag (both Cell Signaling), were used at 1:1000 dilution, β-Actin (Sigma) at 1:4000 dilution, with all incubated at 4 °C overnight. Secondary HRP-linked antibodies (anti-mouse and anti-rabbit, both Cell Signaling) diluted 1:1000 in TBS-T with 1% milk were applied for 1 hr at room temp. Blots were incubated with ECL reagent (Pierce) and developed on film (Sigma) with X-O-Graph developer system.

Cells were transfected as described above, and protein lysates were prepared 72 hours post transfection. The protein concentrations were determined using an amido black assay. Protein samples were separated onto 4–15% Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Biorad). Proteins were then transferred 1 hr onto a polyvinylidene fluoride membrane. That membrane was blocked for 2 h in 5% milk in 1X Tris-buffered saline (TBS) and 0.01% Tween 20 at room temperature. The CjCas9 and SpCas9 protein fused with HA-tag were detected using anti-HA tag (1:1,000; Cell Signaling Technology, Danvers, MA) and β-actin with anti-β-actin (1:1,000; Cell Signaling Technology) antibodies, respectively, and visualized with HRP conjugated anti-rabbit secondary antibody (1:20,000; Dianova, Hamburg, Germany) using West Pico Chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA). The revelation was made at different times of exposition to visualise all possible bands on the western.

Cellular extracts were prepared using lysis buffer containing 50 mM Tris HCL (pH 7.5), 250 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and EDTA free protease inhibitor (Roche 11873580001). Extracts were run on a 4–12% Tris-Glycine gel (BioRad) and transferred onto PVDF membranes. Blots were blocked in 5% milk PBS-T for 1 h at room temperature followed by overnight incubation at 4 °C with primary antibodies at 1:1,000 (anti-HA Tag, Cell Signaling 3724S; anti-Flag Tag, Sigma F1804; anti-DDX5, Bethyl A300-523A; anti-DDX17, Bethyl A300-509A). Horseradish peroxidase (HRP) -conjugated secondary antibodies were used at 1:10,000 (anti-rabbit HRP, Santa Cruz sc-2030) or 1:1,000 (anti-mouse HRP, Cell Signaling 7076S).

The whole-cell lysate was extracted using RIPA buffer, and a western blot was performed following standard protocol. Primary antibodies anti-HA-tag (Cat# 2367) and anti-Bcl-2 (Cat# 4223) were from Cell Signaling Technology, and anti-tubulin (Cat# 12004166) was from BioRad. The results were developed using ChemiDoc MP Imaging System (BioRad) and quantified using Image Lab (BioRad). PowerUp™ SYBR™ Green Master Mix (Thermo Fisher, A25741) was used for qPCR using an Applied Biosystems ViiA 7 real-time PCR system. Primers were included in Supplementary Table S12.

Cells were lysed in 2% SDS buffer (25 mM Tris-Hcl pH 7.5, 100 mM Nacl, 3 mM EDTA, 7% Glycerol) and fresh protease inhibitors. Extracts were sonicated for 10 s and centrifuged at 12,000 × rpm for 10 min to remove cell debris. Western blotting was performed using the following primary antibodies: mouse monoclonal anti-Gapdh 6C5 (#sc32233, Santa Cruz Biotechnology); mouse monoclonal anti-Tubulin B512 (#T5168, Sigma-Aldrich, Milan, Italy); mouse monoclonal anti-b-Actin (ACTBD11B7) (Santa Cruz Biotechnology sc-81178), rabbit polyclonal anti-Golm1 (#PA5-30622, Thermo Scientific); rabbit polyclonal antibodies: anti-Ago2 (#2897, Cell Signaling); rabbit monoclonal antibodies anti-Ago1 (D84G10) XP (#5053, Cell Signaling); rabbit monoclonal antibodies anti HA-Tag (#3724, Cell Signaling).
As secondary antibodies goat anti-mouse and anti-rabbit conjugated to horseradish peroxidase were used (Bethyl Laboratories). Western blot analysis was performed with the aid of the enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). ECL detection was done using a ChemiDoc-It Imaging System (UVP, Upland, CA) instrument.