Phospho sapk jnk thr183 tyr185 p jnk

Muscle tissue were dissected and homogenized in cell lysis buffer (Servicebio, China) added with phosphatase inhibitors (Promotor, China). The lysates were centrifuged at 12,000 rpm for 15 min at 4°C. Supernatant was collected, and protein concentration was determined using bicinchoninic acid assays. Total protein (20–40 μg) was electrophoresed on 8%–10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose filter membranes. Then the membranes were probed with primary antibodies: GRP78/BiP (1:1,000, 3,177, Cell Signaling Technology), p62 (1:1,000, 18420-1-AP, Proteintech), ATG12 (1:1,000, 4,180, Cell Signaling Technology), Beclin-1 (1:1,000, 3,495, Cell Signaling Technology), Phospho-SAPK/JNK (Thr183/Tyr185) (P-JNK) (1:1,000, 4,668, Cell Signaling Technology), JNK1 (1:1,000, 3,708, Cell Signaling Technology), Phospho-eIF2α (P-eIF2α) (1:1,000, 3,398, Cell Signaling Technology), eIF2α (1:1,000, 5,324, Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1: 5,000, 60004-1-Ig, Proteintech). Then the membranes were incubated with horseradish peroxidase-labeled anti-rabbit, or anti-mouse secondary antibody (1:5,000, Cell Signaling Technology). Target protein bands were visualized with chemiluminescence reagents and assessed using Fiji software (NIH).