Nitrocellulose membrane

As was described previously in our publications [51 (link),59 (link)], frozen left ventricular heart tissue was powdered, extracted in SDS lysis buffer (20%, 10 mmol/L EDTA, 100 mmol/L Tris, pH 6.8) and diluted in Laemmli buffer. Proteins were separated in 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (0.2 m pore size, Advantec, Tokyo, Japan). Membranes were blocked in 5% low-fat milk and then incubated with primary antibodies (Table 2) and horseradish peroxidase-linked secondary antibody (Table 2). Enhanced chemiluminescence was used for detection of proteins, which were consequently in triplicate quantitated using Carestream Molecular Imaging Software (version 5.0, Carestream Health, New Haven, CT, USA). For protein normalization GAPDH protein was used.

LFIA was developed over the LFIA designed in previous study²⁶ (link)^,27 (link). Briefly, purified monoclonal antibodies and anti-mouse IgG (Jackson Immuno Research, PA, USA) were diluted in 50 mM Tris buffer) and dropped onto nitrocellulose membrane (ADVANTEC, Tokyo, Japan) to give the test and control line, respectively. The membrane was then dried at 40°C for 30 min to immobilize antibodies. A reagent pad including monoclonal antibodies conjugated with colloidal gold (British Biocell Intonational, Crumlin, UK) was placed upstream of test lines as sample application point. A test strip and two sealed pots containing a reducing reagent (containing 0.47 mol/l Fe(NH₄)₂(SO₄)₂) and a silver-ion reagent (0.31 mol/l AgNO₃) were placed in a cartridge (Figure S7).

Frozen left ventricular tissue was homogenised in lysis buffer of 20% SDS, 10 mmol/L EDTA, 100 mmol/L Tris and pH 6.8. This was diluted in Laemmli buffer. Equal amounts of protein per lane were separated in 10% SDS-polyacrylamide gels at 120 V (Mini-Protean Tetra Cell, Bio-Rad, Hercules, CA, USA). The proteins were transferred to a nitrocellulose membrane (0.2 m pore size, Advantec, Tokyo, Japan) and blocked for 4 h with 5% low-fat milk prior to overnight incubation at 4 °C with the following primary antibodies; anti-Cx43, C6219 Sigma-Aldrich, Saint-Louis, MO, USA; anti-PKCε, sc-214; anti-PKCδ, sc-213; anti-GAPDH, sc-25778—all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. The membranes were washed with Tris-buffered 0.1% Tween (TBS-T) and incubated for 1 h with a horseradish peroxidase-linked secondary antibody (anti-rabbit, diluted 1:2000, Cell Signalling Technology, Danvers, MA, USA, #7074), then washed in TBS-T and visualised by enhanced chemiluminescence. Finally, the Protein bands were quantified by version 5.0 Carestream molecular imaging software densitometric analysis (Carestream Health, New Haven, CT, USA) and normalisation to the GAPDH band. The representative immunoblots (proteins and GAPDH) in figures were spliced to allow better comparison. The spliced blots were from the same gel.

The dss1Δ strain has been described before [23 (link)]. The pDUAL vector [26 (link)] was used for the expression of dss1⁺ and the dss1 variants carrying N-terminal HFG (6His, Flag, green fluorescent protein (GFP)) tags. Cloning and mutagenesis were performed using Genscript. The yeast strains were transformed using lithium acetate [27 (link)]. Growth assays were performed on Edinburgh minimal media (EMM2) (San Diego, CA, USA) as described previously [23 (link)]. The preparation of cell lysate samples for SDS-PAGE was performed using trichloroacetic acid and glass beads as described previously [23 (link)]. The samples were separated by SDS-PAGE on 12.5% acrylamide gels and transferred to 0.2 μm pore-size nitrocellulose membranes (Advantec, Tokyo, Japan). The antibody was anti-GFP (1:1000, Chromotek, Planegg, Germany, Cat# 3H9). Secondary horseradish-peroxidase-conjugated antibodies were from Dako Cytomation. Equal loading was checked using stain-free imaging with 0.5% trichloroethanol (Sigma, St. Louis, MO, USA).

GST-tagged amino-terminal (amino acids 1–275) and carboxy-terminal half (amino acids 271–545) DIDO1 proteins were designated as DIDO1_N and DIDO1_C, respectively, and purified as described above. GST–FOXJ2 and GST–CPSF2 were purchased from Abnova (Taipei, Taiwan). GST and GST fusion proteins (0.3 μg) were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrically transferred onto nitrocellulose membranes (Advantec, Tokyo, Japan). The membranes were blocked using a blocking solution [0.5% skim milk powder in a buffer comprising 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween 20], and the blotted proteins were probed with primary antibodies including anti-GST (goat) (Rockland, Gilbertsville, PA), anti-DIDO1 (rabbit) (Aviva Systems Biology, San Diego, CA), or anti-FOXJ2 (rabbit) (Thermo Fisher Scientific), anti-CPSF2 (rabbit) (GeneTex, Irvine, CA) or from sera from HDs (#30017) or patients with TIA (#07060, #07175, and #07207) or AIS (#07115, #07581, and #07684). After incubation with horseradish peroxidase-conjugated secondary antibodies (anti-goat IgG, anti-rabbit IgG, and antihuman IgG; Santa Cruz Biotechnology, Santa Cruz, CA), immunoreactivity was determined with Immobilon™ Western HRP Substrate (Merck KGaA, Darmstadt, Germany) as previously described [25 (link)–30 (link), 39 (link)–43 (link)].