Anti mouse immunoglobulin g igg

Total proteins were extracted using RIPA buffer containing a protease inhibitor cocktail. After centrifuging at 13,000 rpm for 15 minutes at 4 ℃, the soluble cell extracts were used for immunoblots according to a previous protocol (10 (link)). The primary antibodies included the following: anti-EYA3 (Proteintech, Wuhan, Hubei, China; #21196-1-AP), anti-SIX5 (Invitrogen, Shanghai, China; #PA5-75417), anti-p300 (Santa Cruz Biotechnology, Shanghai, China; sc-48343), anti-GAPDH (Santa Cruz Biotechnology; #sc-47724), anti-Flag (Sigma-Aldrich; #SAB4200071), and anti-Myc (Invitrogen; #R95125). The horseradish peroxidase (HRP)-conjugated secondary antibodies included the following: anti-mouse immunoglobulin G (IgG) (Sigma-Aldrich; #GENA931) and HRP-conjugated anti-rabbit IgG (Sigma-Aldrich; #GENA934).

Immortalized DM1 fibroblasts were seeded at 4.0 × 10⁴ cells per well in a 24-well plate (Falcon). After transfection and 4 days of differentiation, the transdifferentiated myotubes were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and washed three times with 1× PBS. Myotubes were permeabilized with PBS-T (0.3% Triton-X in PBS) and blocked (PBS-T, 0.5% BSA, and 1% donkey serum) for 30 min at RT. They were then incubated with MBNL1 primary antibody (1:200, MB1a [4A8], Developmental Studies Hybridoma Bank⁵² (link)) at 4°C overnight. After three PBS-T washes, the cells were incubated for 1 h with a biotinylated anti-mouse-immunoglobulin G (IgG) (1:200, Sigma-Aldrich) and subsequent Avidin-Biotin amplification (Elite ABC kit, VECTASTAIN) for 30 min at RT. They were followed by three PBS-T washes and incubation with streptavidin-fluorescein isothiocyanate (FITC) fluorophore (1:200, Vector) for 2 h at RT. After three washes with PBS, the cells were mounted with VECTASHIELD mounting medium containing 2 μg/mL DAPI (Vector) to detect the nuclei.
Images of DM1 cells were taken on an Olympus FluoView FV100 confocal microscope. The images were taken at a 40× magnification and quantified using ImageJ with the following formula at a threshold of 10: mean pixel intensity = gray value/area.