Psapk jnk thr183 tyr185

Western blot analysis for erythropoietin (EPO) (1:500, Abcam), STAT5a,b (1:600), and Bcl-XL (1:1000), SAPK/JNK (1:1000, Cell Signaling), and phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (1:11,000, Cell Signaling) was performed using 19 μg protein extract from naïve mice and mice with 1 and 6 h survival after SCI. Proteins were separated on 4-12% SDS-PAGE gels (Invitrogen) using MOPS SDS (Invitrogen) containing 0.25% antioxidant (Invitrogen) as previously described [13 (link)]. β-actin (1:100,000, Sigma-Aldrich) or transcription factor II B (TFIIB) (1:1000, Cell Signaling, Leiden, The Netherlands) were used as loading controls. SeeBlue Plus2 Prestained standard (Invitrogen) was used as a molecular marker. Bands were quantified using Image Lab Software (Bio-Rad, Copenhagen, Denmark).
The same naïve Tnf^fl/fl mice were included on all gels and data were normalized according to protein concentrations in individual plots. Analysis was performed on unmerged blots with the same exposure time using Image Lab (Bio-Rad). Analysis was performed with n = 5 mice/group and data were normalized to the loading control and presented as percentages relative to naïve Tnf^fl/fl mice.

Preparation of whole cell lysates and western blotting analysis was performed as previously described.³⁴, ³⁵ Primary antibodies against cyclin A2 (1:1000), cyclin B1 (1:1000), cyclin D1 (1:1500), cyclin D2 (1:1500), cyclin D3 (1:1500), cyclin E1 (1:1000), CDK2 (1:1500), CDK4 (1:1500), CDK6 (1:1500), CDC2 (1:1500), β‐catenin (1:1500), GSK‐3β (1:2000), p‐GSK‐3β (Ser 9) (1:2000), p‐AKT (Ser 473) (1:2000), c‐jun (1:1500), c‐myc (1:1500), PCNA (1:1500), PARP (1:1500), cleaved‐PARP (1:1500), pro‐caspase 3 (1:1500), cleaved‐caspase 3 (1:1500), RIPK 1 (1:1500), p‐RIPK 1 (Ser166) (1:1500), ERK 1/2 (1:2000), p‐ERK (Thr202/Tyr204) (1:2000), p‐SAPK/JNK (Thr183/Tyr185) (1:1500) and p‐p38 MAPK (Thr180/Tyr182) (1:2000) were purchased from Cell Signalling Technology (Danvers, MA). An antibody against AKT (1:500) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibodies against β‐actin (1:2000) and β‐tubulin (1:2000) were acquired from Beijing Ray Antibody Biotech (Beijing, China). The grey values of proteins expression in Western blotting were quantified using the Image J software (NIH Image, Bethesda, MD).

Protein extracts were resolved by 8% to 15% SDS-PAGE. The proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes; the membranes were blocked with 5% nonfat dry milk and incubated with primary antibodies. Antibodies against c-Myc (3G32), p-STAT1 (Tyr701), ERK1 (K-23), JNK (FL), MEK1/2, p38 (H-147), p-MEK-1 (Thr291), p-ERK1/2 (Thr204), and GAPDH (FL-335) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PU.1, p21 waf1/CIP1, p-STAT1 (Ser727), p-p38, MAPK (Thr180/Tyr182), p27/Kip1 (D37H1), STAT1, C/EBPβ (LAP), and p-SAPK/JNK (Thr183/Tyr185) antibodies were purchased from Cell Signaling Technology (Danvers, MA). The western blot was visualized using HRP-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc., West Grove, PA), followed by enhanced chemiluminescence detection (Biological Industries, BeitHaemek, Israel).

Hepatic expression of proteins implicated in fibrosis, apoptosis, mitochondrial function, and oxidative stress was assessed by western blotting [20 (link)]. Each membrane was blotted with the respective primary antibody [p-SAPK/JNK (Thr183/Tyr185) (#9251, Cell Signaling Technology, USA), Bax (#2772, Cell Signaling Technology, USA), Bcl-2 (#2876, Cell Signaling Technology, MA, USA), Total OXPHOS (ab110413, Abcam, UK), Citrate Synthase (CS; Cat. 16131-1-AP, Proteintech, UK), Catalase (ab1877, Abcam, UK), MnSOD (06-984, Merck Millipore, UK), p-AMPKα (Thr172) (#2535, Cell Signaling Technology, USA), and AMPKα1 (ab3759, Abcam, UK)]. Proteins were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (Jackson Immuno Research, UK). Detection was performed with Super Signal West Pico Chemiluminescent substrate (Thermo Scientific, UK) using an ImageQuant^TM LAS 4000 (GE Healthcare Life Sciences, UK). Band intensities were quantified by optical densitometry (Scion Image-Release Beta 3b, NIH, USA). To confirm equal protein loading, membranes were stripped (Restore™ Western Blot Stripping Buffer, Thermo Scientific, UK) and reblotted with an anti-GAPDH antibody (#2118, Cell Signaling Technology, USA). Western blotting data were calculated relative to the CC group, which were defined as 100 percent.