Ab40567

Immunostaining of muscle biopsy samples was performed as previously described [31 (link)]. Briefly, muscle sections were thawed at room temperature and then fixed in 100% cold acetone for 10 min. In order to reveal only the proteins trapped in aggregates, the muscle sections were then incubated in 1 M potassium chloride (KCl) to remove all soluble proteins. Primary antibodies against PABPN1 (ab75855 Abcam; 1:100), HSP70 (MA1-90,504 Invitrogen; 1:100), PRMT1 (ab73246 Abcam; 1:100), dystrophin (NCL-dys1 Novocastra; 1:10), laminA/C (ab40567 Abcam; 1:100), p62/SQSTM1 (MBL PM045 1:200), RPL24 (GTX114729 1:100), GRP78/BiP (CST3177 Cell signaling 1:200) were then incubated overnight at 4 °C. When necessary a directly conjugated PABPN1-488 antibody (ab206056 Abcam) was used. The next day, the muscle sections were rinsed and incubated with the appropriated fluorescent secondary antibodies prior to counterstaining nuclei with Hoechst. For the eMyHC, spectrin and laminA/C staining, slides were thawed at RT and blocked in PBS containing 2% FBS for 30 min at RT. Primary antibodies for embryonic myosin (F1.652 DSHB; 1:4), human spectrin (NCL-SPEC Novocastra; 1:50) human laminA/C (ab40567 Abcam, 1:300) and laminin (Z0097 Dako; 1:400) were incubated for 1 h at RT, sections were rinsed and incubated with appropriate secondary antibodies prior to counterstaining with Hoechst.

Frozen muscles were sectioned at 8 μm thicknesses using a Leica CM1950 cryostat maintained at −20℃. The muscle sections were first stained with hematoxylin and eosin (H/E) for histological assessment of xenograft regeneration. Muscle sections were also immunostained with human‐specific spectrin (SPEC1‐CE, 1:50, Leica) and human‐specific lamin A/C (ab40567, 1:200, Abcam), while total muscle tissue was marked by 4′6‐diamidino‐2‐phenylindole (DAPI) staining (outside human‐marked tissue) or by immunostaining with fluorescently conjugated wheat germ agglutinin (WGA alexafluor‐647, W32466, 1:500, Thermo Fisher Scientific). Specifically, muscle sections were fixed in ice‐cold methanol for 10 minutes, washed in phosphate‐buffered saline (PBS), and blocked in PBS supplemented with 2% goat serum (GTX73249, GeneTex) and mouse‐on‐mouse (M.O.M.) blocking reagent (MKB‐2213, Vector Laboratories). Muscle sections were incubated with primary antibodies overnight at 4℃ and subsequently probed with Alexa Fluor secondary antibodies (Life Technologies). Finally, muscle sections were mounted with Prolong Gold Mounting Media (Life Technologies) with DAPI for nuclear staining.

The primary antibodies used were as follows: rabbit anti-INPP5E (17797-1-AP; Proteintech); rabbit anti-INPP5E (HPA065758; Sigma), rabbit anti-DDK (14793S; Cell Signaling); rabbit antipericentrin (ab4448; Abcam); mouse anti-PLK1 (ab17056; Abcam); mouse anti-gamma-tubulin (GTU-88; Sigma); mouse phospho-Aurora (2914S; Cell Signaling); mouse anti-alpha-tubulin (A11126; Life Technologies); mouse anti-PI(4,5)P₂ (Z-P045; Echelon Biosciences), rabbit anti-CENPA (2186S; Cell Signaling); mouse antiactin (A5441; Sigma); mouse anti-GAPDH (sc-365062; Santa Cruz Biotechnology), anti-phospho-H3 (9701L; Cell Signaling), and anti-cyclin B1 (4135S; Cell Signaling); and mouse anti-lamin A+C (ab40567; Abcam).