Mouse anti flag tag

For immunostaining, HEK293T or HeLa cells grown on coverslips were fixed with 4% PFA in PBS at room temperature for 15 min and permeabilized with 0.25% PBST (PBS containing 0.25% Triton X–100) at room temperature for 15 min. Cells were blocked in 5% normal goat serum for 1 hr at room temperature. The samples were incubated overnight at 4°C with primary antibodies: rabbit anti-HA-tag (1:500; Cell Signaling Technology, #3724) and mouse anti-FLAG-tag (1:500; Sigma, F9291). Cells were washed in PBST and Alexa Fluor 488-anti-rabbit (1:200; Jackson Immunoresearch, 111545144) and Cy3-anti-mouse (1:200; Jackson Immunoresearch, 115165003) were used as the secondary antibodies. The samples were then stained with DAPI (1 μg/mL, Sigma, D9542) for 5 min at room temperature, washed, and mounted with Fluoromount aqueous mounting medium (Sigma, F4680).

For western blotting, cells were lysed with RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH8.0, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) containing EDTA-free protease inhibitor cocktail (Roche). After centrifugation, clear supernatant was collected, added with protein sample buffer to final concentration of 50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 30 mM DTT, 0.002% bromophenol blue, boiled for 10 min and separated on SDS-PAGE gel. Proteins were semi-dry transferred onto PVDF membrane, blocked with 5% skim milk and probed with antibodies as indicated. For immunostaining, cells seeded on chamber-slides were fixed with 4% paraformaldehyde, 0.5% NP-40 permeabilized and blocked with 5% normal donkey serum (Jackson ImmunoResearch), followed by staining using the indicated antibodies. Images were captured using LSM880 confocal microscope (ZEISS). Mouse anti-FLAG-tag (Sigma-Aldrich), anti-beta-actin (Sigma-Aldrich), anti-GAPDH (Santa Cruz Biotechnology) and rabbit anti-HA-tag (Cell Signaling Technology), anti-IRF3 (Santa Cruz Biotechnology) antibodies were used.

Antibodies used in the study included the following: rabbit anti-YTHDC1 (GTX32976, GeneTex, USA); rabbit anti-YTHDF2 (24744-1-AP, proteintech); rabbit anti-METTL3 (15073-1-AP, proteintech); mouse anti-Flag-tag (F1804, SigmaAldrich, Saint Louis, MO, USA); mouse anti-HA-tag (M180-3, MBL, Japan); mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CB100127, California Bioscience, Coachella, CA, USA); rabbit anti-IAV NP, NS1, and NEP, (GTX125989, GTX125990, and GTX125953, GeneTex, USA); mouse anti-NP (produced in our laboratory); control rabbit IgG polyclonal (AC005, ABclonal Biotechnology, Cambridge, MA, USA); horseradish peroxidase-conjugated anti-mouse and anti-rabbit (BF03001 and BF03008, Beijing Biodragon Inmmunotechnologies, China); Alexa Fluor 594-conjugated goat anti-rabbit (GR200G-43C, Sungene Biotech); fluorescein isothiocyanate (FITC) goat anti-mouse (GM200G-02C, Sungene Biotech); and FITC-goat anti-rabbit (GR200G-02C, Sungene Biotech). 4′,6′-diamidino-2-phenylindole (1:1000) (no. C1002) was purchased from the Beyotime, China.

Infected or transfected cells were harvested and lysed in cold TNE buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 2 mM EDTA [pH 8.0], 0.1% 2-mercaptoethanol and protease inhibitor cocktail). After incubation on ice for 30 min, cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C. The clarified supernatant was mixed with 5XSDS-PAGE loading buffer, boiled at 100°C for 10 min and then subjected to 12% sodium dodecyl sulfate-polyacrylamide gel. The primary antibodies used were as follows: mouse anti-HPIV3 (1:2500, Abcam), rabbit anti-β-actin (1:1000, Proteintech), mouse anti-Myc tag (1:2500, Santa Cruz), mouse anti-HA tag (1:10000, Sigma), mouse anti-Flag tag (1:2500, Sigma), mouse anti-cofilin (1:2500, Proteintech), and rabbit anti-p-cofilin (1:1000, Cell Signaling Technology). HRP-conjugated goat anti-mouse IgG (1:5000, Sigma) and HRP-conjugated goat anti-rabbit IgG (1:5000, Sigma) were used as secondary antibodies.

Tissue fragments were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS using ball bearings and a TissueLyser (Qiagen). Lysis buffer was supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher). Protein lysates were quantitated using Pierce BCA protein assay (Thermo Fisher). Western blotting was performed using Miniprotean TGx 4–20% SDS-PAGE gels, Tetra Cell rig, and Transblot Turbo transfer system (Biorad). Membranes were blocked with 5% milk in 1× TBS-T, before overnight incubation with primary antibodies. HRP-conjugated secondary antibodies against rabbit- and mouse-derived primary antibodies were used at 5000-fold dilution and were obtained from Cell Signaling Technologies (7074S, 7076S). Chemiluminescence was visualized and recorded digitally using the Chemidoc XRS + Imaging System (Biorad). Primary antibodies used in this study include: rabbit anti-Cdkal1 (abcam, ab68045), mouse anti-ANT1 (abcam, 110322), rabbit anti-MMS19 (Proteintech, 16015-1-AP), mouse anti-total OXPHOS rodent cocktail (abcam, ab110413), mouse anti-FAM96B (Santa Cruz, sc-376801), mouse anti-FLAG tag (Sigma, F1804), mouse anti-FLAG M2 HRP-conjugated (Sigma, A8592), rabbit anti-UCP1 (abcam, 23841), and rabbit anti-β-Tubulin (Cell Signaling, 2146).

Flag-tagged thin-filament protein localization was imaged in 48-h transduced cardiomyocytes immobilized in two-well Falcon-chambered cell-culture slides (Fisher scientific) precoated with 40 μg/mL laminin (BD Bioscience) at room temperature for 4 h. Cells were fixed with 4% methanol-free paraformaldehyde (TAAB) for 15 min at room temperature; cells were then skinned and slide blocked simultaneously for 1 h using PBS containing 0.1% Triton X-100 and 5% BSA. Cells were incubated overnight at 4°C with mouse anti-FLAG-tag (1:8; Sigma) and rabbit anti-α-actinin (Sigma; 1:500), followed by a further overnight incubation with goat anti-mouse IgG Alexa 564 (1:200; Life Technologies) and goat anti-rabbit IgG Alexa 633 (1:200; Life Technologies). Slides were mounted with slow-fade diamond with DAPI and a 1.5 thickness coverslip and visualized using a Leica TCS SP5 X confocal microscope with a 63× oil immersion objective.
For imaging of NFATc3 following chronic pacing (4 h), cells were immobilized, fixed, and skinned in a round coverslip-bottomed 35-mm culture dish (Matek) using the same conditions as above. Cells were stained with consecutive overnight incubations with first NFAT-c3 (1/50; Santa Cruz) primary and then goat anti-mouse IgG Alexa 568 (1/200; Life Technologies). Finally, the sides of the coverslip were removed using a heated metal scalpel and mounted and imaged as before.

Mouse anti-Gephyrin (1:1000, clones mAb7a, Synaptic Systems #147021), rabbit anti-SUMO-1 (1:250, Epitomics#1563-1), mouse anti-SUMO-1 (1:100, SantaCruz#sc-5308), rabbit anti-SUMO-2/3 (1:250, Cell signaling #4974), rabbit anti-SUMO-2/3 (1:250, Epitomics #2970-1), mouse anti-PIAS-3 (1:500, Sigma #P0117), rabbit anti-vGAT (1:2000, Synaptic Systems #131011); mouse anti-Myc tag (1:5000,Roche #11667149001), rabbit anti-Myc tag (1:5000, Cell Signaling #2278S), and mouse anti-FLAG tag (1:5000, Sigma Aldrich #F3165). All the secondary antibodies were from Jackson ImmunoResearch: Goat anti-Mouse Cy3 IgG (1:500, #115165), Goat anti-Mouse IgG Cy5 (1:500, #115175), Goat anti-Rabbit IgG Cy3 (1:500, #111165), and Goat anti-Rabbit IgG Cy5 (1:500, #111175).