Ab73246

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.

Western blot assay was performed as previously described [16 (link)]. Briefly, proteins isolated from the kidneys of mice were quantified using a BCA kit (Beyotime, Shanghai, China). The proteins were then separated by SDS/PAGE and transferred onto poly(vinylidene difluoride) membranes (Millipore, Billerica, MA, USA), which were subsequently incubated overnight at 4 °C with the primary antibodies (dilution 1 : 1000). Antibodies against NGAL (ab216462), PRMT1 (ab73246), GAPDH (ab9483), Smad3 (ab84177), p‐Smad3 (ab52903), E‐cadherin (E‐cad) (ab231303), pro caspase‐3 (ab32499), COX‐2 (ab179800), p‐STAT3 (ab76315), and STAT3 (ab68153) were purchased from Abcam (Cambridge, UK). Antibody against sIL‐6R (23457) was purchased from Proteintech (Rosemont, IL, USA). Antibody against cleaved caspase‐3 (SAB4503292) was purchased from Sigma‐Aldrich. The membranes were then incubated with the secondary antibody coupled with horseradish peroxidase at room temperature for 30 min. The bands of proteins were visualized via enhanced chemiluminescence substrate (Solarbio, Beijing, China). GAPDH served as an internal control.