Pa5 64821

The proteins of H-ASCs and AAA-ASCs were extracted and their concentration determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, 231227). A total of 25 μg of protein of each sample was loaded, separated by SDS/PAGE, and then transferred to PVDF membranes. After blocking with 5% fat-free milk in TBST, the membranes were incubated at 4°C overnight with the following antibodies: anti-p-Drp1 ser616 (1 : 1000, Invitrogen, PA5-64821), anti-Drp1 (1 : 1000, Invitrogen, PA5-20176), anti-Mfn2 (1 : 1000, Abcam, ab124773), anti-p53 (1 : 1000, Abcam, ab26), anti-p21 (1 : 1000, Abcam, ab109199), anti-Beclin (1 : 1000, CST, 3738), anti-LC3I/II (1 : 1000, CST, 4108), anti-p62 (1 : 1000, CST, 5114), and GAPDH (1 : 1000, CST, 2118). Subsequently, after washing with TBST three times, the membranes were incubated with secondary antibodies (1 : 3000, CST) at room temperature for 1 hour and then exposed in a dark room.

MSCs were cultured under normoxia or SD/H conditions as described above. In indicated groups, 10 μM U0126 (HY-12031A, MCE) or 10 μM ML221 (HY-103254, MCE) was added together with Apelin-13. The protein of MSCs with different treatments was extracted using RIPA buffer (9806, CST), and the concentration of each sample was determined using a bicinchoninic acid assay kit (231227, Thermo). A total of 30 μg protein from each sample was separated on SDS-PAGE gel and transferred to PVDF membranes. After washing with Tris-buffered saline containing 0.1% Tween-20 (TBST) three times, the membranes were blocked by TBST with 5% fat-free milk and then incubated overnight at 4°C with the following primary antibodies: anti-p-ERK (9101, CST), anti-t-ERK (4695, CST), anti-Drp1 (PA5-20176; Invitrogen), anti-p-Drp1 ser616 (PA5-64821, Invitrogen), anti-Mfn1 (ab57602, Abcam), anti-Mfn2 (ab124773, Abcam), anti-APJ (20341-1-AP, Proteintech), and anti-GAPDH (2118, CST). Subsequently, the membranes were washed with TBST three times and incubated with horseradish peroxide-conjugated secondary antibodies at room temperature for 1 hour. Finally, the membranes were exposed by enhanced chemiluminescence (ECL plus) (Amersham) in a dark room.

The protein of each sample was extracted using RIPA buffer (9806, CST), and then the amount of concentrated protein was measured. The proteins were separated on SDS‐PAGE gel, transferred to PVDF membranes and then washed with Tris‐buffered saline (TBS) with 0.1% Tween‐20 (TBST). After blocking with 5% fat‐free milk in TBS, the membranes were incubated with the following primary antibodies: anti‐p‐Drp1 ser616 (PA5‐64821; Invitrogen), anti‐Drp1 (PA5‐20176; Invitrogen), anti‐Mfn2 (ab124773; Abcam) and GAPDH (2118, CST) at 4°C overnight. Then, the membranes were incubated with horseradish peroxide‐conjugated secondary antibodies for 1 hour at room temperature. Finally, the membranes were exposed using enhanced chemiluminescence (ECL plus) (Amersham).