Protein kinase b akt

Total proteins were extracted from homogenized tissues and cell lysates using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, Rockford, USA). An equal amount of protein lysates was separated on 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After an overnight incubation with the following primary antibodies at 4 °C: HOXB5 (1:1000, #109375, Abcam, Cambridge, UK), SESN3 (1:800, #97792, Abcam), IGF1R (0.1 μg/mL, #AF-305-SP, R&D Systems, Inc., Minneapolis, USA), protein kinase B (Akt, 1:1000, #9272, Cell Signaling), phosphorylated Akt (p-Akt, 1:1000, #9271, Cell Signaling, Danvers, USA), PCNA (PCNA, 1:1000, #18197, Abcam), p27 (1:1000, #32034, Abcam), B-cell lymphoma 2 (Bcl-2, 1:800, #59348, Abcam), E-cadherin (1:2000, #40772, Abcam), N-cadherin (1:1000, #76057, Abcam), cleaved caspase-3 (1:1000, #49822, Abcam), and GAPDH (1:2000, #37168, Abcam), the membranes were washed with Tris-Buffered Saline and stained with secondary antibody (1:2000, #6721, Abcam) for 60 min at room temperature. The images were captured using Alphalmager™ 2000 Imaging System (Alpha Innotech, USA).

Rats were sacrificed 4 weeks after burn injuries by an overdose of Zoletil 50. Lumbar 3,4,5 (L345) spinal cords were collected, and the dorsal horn area and skin were separated, frozen in liquid nitrogen, and stored at -80°C. L345 dorsal horn specimens and skin were homogenized in an ice-cold lysis buffer, T-PER Tissue Protein Extraction Reagent (Thermo Scientific) with one tablet of Complete Protease Inhibitor Cocktail (Roche) per 25 mL and then centrifuged (15,000 g) for 30 minutes at 4°C. Each protein concentration in the supernatant was measured using bovine serum albumin as the standard. The procedures and analyses of Western blots were performed through the same method as our previous report [26 (link)]. The primary antibodies of COX-2 (1:1000, Cell Signaling, Danvers, MA), iNOS (1:1000, Abcam, Cambridge, MA), and nNOS (1:1000, Abcam, Cambridge, MA), protein kinase B (AKT) (1:1000, Cell Signaling, Boston, MA), p-AKT (1:1000, Cell Signaling, Boston, MA), Bcl-2 Associated X protein (Bax) (1:1000, Proteintech Group, Chicago, IL), Bcl-2 (1:1000, Abcam, Cambridge, MA), and β-actin (1:20000 dilution, Sigma-Aldrich, Saint Louis, MO) were used in this study.

Jejunal and colonic tissues were ground in liquid nitrogen and lysed using radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors. After sonication and centrifugation, protein concentration in the supernatant was quantified using a bicinchoninic acid (BCA) protein assay kit (02912E, CWbiotech, Beijing, China). Supernatant with 30 μg proteins from each sample was separated in sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μm, Millipore, United States). Membranes were blocked and incubated with the primary antibodies of phosphatidylinositol 3-kinase (PI3K, #4257, Cell Signaling Technology, Danvers, MA, United States), protein kinase B (Akt, #9272, Cell Signaling Technology), phosphor-Akt (p-Akt, #9271, Cell Signaling Technology), and β-actin (#4970, Cell Signaling Technology) overnight at 4°C. The membranes were incubated with secondary antibodies (111-035-003, Jackson, United States) for 1 h at room temperature and reacted with electrochemiluminescence (ECL, WBKLS0500, Millipore, United States). The intensity of protein bands was analyzed using the ImageJ software.

Western blots were performed as described previously [29 (link)] for glycogen synthase kinase 3 beta (GSK3β; Abcam, Cambridge, UK), phospho-GSK3β Ser9 (Abcam), glycogen synthase 1 (GS; Abcam), phospho-GS Ser641 (Abcam), β-actin (Abcam), protein kinase B (Akt; Cell Signaling Technology, Beverly, MA, USA), phospho-Akt Ser473 (CST), insulin receptor substrate 1 (IRS-1; CST), phospho-IRS-1 Ser307 (CST), phospho-IRS-1 Y632 (CST), forkhead box protein O1 (FoxO1; CST) and phospho-FoxO1 Thr24 (CST) [30 (link),31 (link)].

Fetal bovine serum (04‐001‐1ACS; Biological Industries, Shanghai, China), lentiviral (GenePharma, China), SYBR Green Master Mix (Q121–02‐AA; Vazyme), Lipofectamine 3000 (L3000015; Invitrogen, CA, USA), RNA‐Quick Purification Kit (RN001; Yishan Biotechnology), PrimeScript™ RT Master Mix kit (RR036A; Takara), PE anti‐B7‐H3 Ig (331606; BioLegend, CA, USA), FITC anti‐B7‐H3 Ig (FAB1027F; R&D), B7‐H3 mouse monoclonal antibody (mAb; 66481‐1‐Ig; Proteintech), FN mouse mAb (66042‐1‐Ig; Proteintech, CA, USA), E‐cadherin mouse mAb (14472; Cell Signaling Technology, Boston, MA, USA), N‐cadherin Rabbit mAb (13116; Cell Signaling Technology), matrix metallopeptidase 9 (MMP9) Rabbit mAb (ab76003; Abcam, Milton, Cambridge, UK), phosphoinositide 3‐kinase (PI3K; 4249; Cell Signaling Technology), phosphorylated (p)‐PI3K (17366; Cell Signaling Technology), protein kinase B (AKT; 4691; Cell Signaling Technology), p‐AKT (4060, Cell Signaling Technology), extracellular regulated protein kinase (ERK; 4695; Cell Signaling Technology), p‐ERK (4370; Cell Signaling Technology), p38 (8690; Cell Signaling Technology), p‐p38 (4511; Cell Signaling Technology), protein A + G agarose (P2055; Beyotime), B7‐H3 Ig (14058; Cell Signaling, USA) were used in this study.