Nbp1 89300

Cells were trypsinized, pelleted by centrifugation at 300 g for 5 min and lysed in RIPA buffer, containing 50 mM NaCl, 25mM Tris-HCl pH8.0, 0.1% SDS. Lysates were centrifuged at 16,000 g and Laemmli buffer was added to supernatant and further incubated for 5 min at 100 °C. Protein lysates were run in 6% polyacrylamide gel and transferred to 0.45 µm nitrocellulose membrane. Membranes were blocked with 5% milk in PBS with 0.05% Tween-20 (PBS-T) (Sigma Aldrich, St. Louis, MO, USA) for 30 min and incubated with an anti-FLNC primary antibody (NBP1-89300, Novus Biologicals, Littleton, CO, USA) or anti-β-actin primary antibodies (AC-15, ab6276, Abcam, Cambridge, UK) for 16 h. Membranes were washed 3 times in PBS-T and incubated with secondary antibodies (Immun-Star Goat Anti-Rabbit/Mouse (GAR/GAM) HRP Conjugate, Bio-Rad Hercules, CA, USA), 1:10000 dilution in 5% milk for 1 h. Chemiluminescence was detected after application of SuperSignal West Femto substrate (Thermo Fisher Scientific, Waltham, MA, USA). Images were analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Human cardiac myocytes (PromoCell) were studied, at 60%–70% confluence, after a minimum of 21 days and after confirmation of GATA4 and ACTC1 expression by qRT-PCR. Cells were treated with 500 IU/mL recombinant IFN-β for 48 hours (R&D Systems). For knockdown experiments, cells were incubated with a mixture of siRNA directed against ISG15 or negative control siRNA (MilliporeSigma) at 50 nM concentration and Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) for 6 hours before incubation with 500 IU/mL IFN-β for 48 hours. Coimmunoprecipitation was performed using Protein G magnetic beads (Cell Signaling Technology) with either an anti–filamin-C antibody (1:50; 86972, Cell Signaling Technology) or isotype IgG (1:50; ab172730, Abcam) before immunoblotting for ISG15 (1:1,000; PA5-88262, Thermo Fisher Scientific) or filamin-C (1:1,000; NBP1-89300, Novus Biologicals).

Immunoblotting was performed with antibodies against the following proteins: ISG15 (1:1,000; mouse, 703132, clone 1H9L21; human and rat, PA5-88262; Thermo Fisher Scientific), GAPDH (1:1,000; 2188, Cell Signaling Technology), cGAS (1:1,000; 31659, Cell Signaling Technology), STING (1:1,000; 13647, Cell Signaling Technology), RIG-I (1:1,000; 3743, Cell Signaling Technology), MAVS (1:1,000; 4983, Cell Signaling Technology), β-actin (1:10,000; A1978, MilliporeSigma), filamin-C (1:1,000; NBP1-89300, Novus Biologicals), vinculin (1:1,000; 4650, Cell Signaling Technology), p62 (1:1,000; 109012, Abcam), and LC3 (1:1,000; 12741, Cell Signaling Technology). Immunoblotting for the soluble and insoluble fractions of filamin-C was performed in mouse hearts as previously described (82 (link)). Densitometry was performed using ImageJ version 1.39 (NIH).