Ab9904

Antibody against QKI was purchased from Santa Cruz Biotech (sc-103851), USA. Antibodies against QKI5, QKI6, QKI7 were from Millipore (AB9904, AB9906, AB9908), USA. Antibody against Smooth Muscle Myosin Heavy Chain (SMMHC) was from AbD Serotec (Rabbit, AHP1117). Antibodies against SM22 (rabbit, Ab14106) and calponin (rabbit, Ab46794) were from Abcam, UK. Antibodies against GAPDH (mouse) and monoclonal anti-α smooth muscle actin (SMA) (Clone 1A4, A5228) were from Sigma. All secondary antibodies were from Santa Cruz Biotech.

Sections from the placenta were immediately fixed with formalin and embedded with paraffin. Sections of 5 μm thickness were then deparaffinized with ethanol following hydration. The sections were then washed in Tris‐buffered saline and further incubated in sodium citrate buffer containing 3% hydrogen peroxide and 50% methanol for antigen exposure. The slides were blocked using 5% blocking serum for 30 min and stained with biotin‐conjugated primary antibody against QKI5 (1:500, AB9904, Millipore) or NRP1 (1:1000, ab81321, Abcam) overnight at 4°C. Streptavidin‐conjugated secondary antibody was added along with haematoxylin for counterstaining.

Protein expression was analysed by harvesting cells in RIPA lysis buffer containing freshly added 1% phenylmethylsulfonyl fluoride for inhibiting proteases. The total protein content of cell lysates was estimated using the standard bicinchoninic acid (BCA) method (Abcam). Forty μg of protein per sample was denatured and loaded onto a 10% SDS‐PAGE gel and subjected to electrophoresis at 100 V for 1.5 h. The separated protein bands were transferred to a polyvinylidene difluoride membrane by semi‐dry blotting and probed with primary antibodies against QKI5 (anti‐human QKI5, 1:1,000, AB9904, Millipore), NRP1 (anti‐human NRP1, 1:1,000, ab81321, Abcam) or MMP9 (anti‐human MMP9, 1:2,000, ab38898, Abcam). β‐actin (anti‐human β‐actin, 1:1,000, ab8226, Abcam) or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (anti‐human GAPDH, 1:3,000, ab181602, Abcam) were used as loading controls. Membranes were incubated with primary antibodies overnight at 4°C and further incubated with the corresponding horseradish peroxidase‐conjugated secondary antibodies, goat anti‐rabbit IgG‐HRP (1:10 000, ab6721) for 1 h at room temperature. Immunodetection of proteins using chemiluminescence was performed with Image‐Pro Plus 6.0 (Media Cybernetics).

Proteins from total cell lysate (150 mM NaCl, 50 mM Hepes pH 7.4, and 1% Triton X-100) were separated by SDS–PAGE and transferred onto nitrocellulose membranes using the Trans-Blot turbo transfer system (Bio-Rad). Membranes were stained with Ponceau Red to confirm equal loading, and then blotted with the primary antibodies against myosin heavy chain (MF-20; DSHB), QKI-5 (AB9904, AB9906, respectively; MilliporeSigma) overnight at 4°C. After three washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies (Sigma-Aldrich) for 45 min and visualized on X-ray films with Western Lightning Plus ECL (PerkinElmer).

Cultured myofibers were fixed in 2% PFA at the indicated time points and washed twice with 1× PBS. Fixed myofibers were then permeabilized with 0.2% Triton X-100, 0.125 M glycine in 1× PBS for 15 min at RT. Blocking followed for 1 h at RT with 2% BSA, 5% horse serum and 0.1% Triton X-100. Primary antibodies were diluted in blocking buffer to detect Pax7 (1:100; Developmental Studies Hybridoma Bank [DSHB]), MyoD (sc-304; Santa Cruz Biotechnology), Myogenin (F5D; DSHB), Dystrophin (MANDRA1[7A10]; DSHB), QKI-5 (AB9904, AB9906, respectively; MilliporeSigma). After 16-h incubation at 4°C, fibers were washed three times with 1× PBS for 10 min. Secondary antibody (AlexaFluor anti-mouse or anti-rabbit 488 or 568 nm), or AlexaFluor-647–conjugated primary antibody against Itga7 (FAB3518R; R & D Systems) was used at a dilution of 1:400 in blocking buffer for 1 h in the dark at RT. Myofibers were washed three times for 10 min with 1× PBS. Finally, the myofibers were transferred to a microscope slide outlined using an ImmEdge hydrophobic barrier pen (H-4000; Vector Laboratories) and mounted with ProLong Gold Antifade Mountant with DAPI (P36935; Thermo Fisher Scientific). Fiber-associated MuSCs were then visualized on a Zeiss Axio Imager M1 microscope (Carl Zeiss) or LSM800 Airyscan confocal microscope and resulting images were analyzed using Zeiss’ ZEN Digital imaging suite software.