P pi3k

At 28 days following the operation, the sciatic nerve tissues containing the area of crush injury site (5 mm away from the sciatic notch) were harvested and fixed with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue were prepared. Moreover, the sections were stained with NF200 (CST, 1:200), S100β (Abcam, 1:200), TuJ1 (Abcam, 1:200), MBP (Abcam, 1:200), CD31 (Abcam, 1:200), CD34 (Abcam, 1:200), VEGFR (Abcam, 1:200), GAPDH (Abcam, 1:200), Akt (Abcam, 1:500), p-AKT (Abcam, 1:500), PI3K (Abcam, 1:500), p-PI3K (ThermoFisher, 1:500) and PTEN (Abcam, 1:500). Secondary antibodies were as follows:Alexa Fluor568–conjugated Goat Anti-Rabbit IgG (Abcam), CoraLite594-conjugated Goat Anti-Mouse IgG (Proteintech, China), CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech), CY3-labeled goat anti-rabbit (Servicebio) and AlexaFluor594-labeled goat anti-rabbit IgG (Abcam). Besides, we also used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence intensity. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy. The percentages of the markers positive areas were calculated by dividing integrated option density by selected region area, then multiplied by 100%. All parameters were measured using ImageJ.

Total protein in the cells was extracted using radioimmunoprecipitation assay buffer (R0010, Solarbio, Beijing, China) and quantified using a protein bicinchoninic acid analysis kit (71285-3, Sigma). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (3010040001, Millipore, Billerica, MA, USA). After being sealed in 5% skim milk for 120 min at room temperature, the membranes were treated with specific primary antibodies overnight at 4°C and with the secondary antibody for 120 min at room temperature. Protein bands were detected with enhanced chemiluminescence solution (PE0010, Solarbio) and imaged with a GelDoc Go system (Bio-Rad, Hercules, CA, USA). Relative expression of proteins was measured using ImageJ with GAPDH as an internal reference. The antibodies used in the experiments are as follows: primary antibodies were FOXA1 (1 : 1000, ab170933, Abcam, Cambridge, MA, USA), SIX4 (1 : 2000, ab176713, Abcam), GAPDH (1 : 2000, GTX124502, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), p-PI3K (1 : 1000, PA5-118549, Thermo Fisher Scientific), PI3K (1 : 1000, #4257S, Cell Signaling Technologies, Beverly, MA, USA), p-AKT (1 : 1000, ab38449, Abcam), and AKT (1 : 500, ab8805, Abcam); secondary antibody was goat anti-rabbit IgG antibody (ab6721, 1 : 2000, Abcam).

Thidiazuron and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reagent for the MTT assay were attained from Sigma‐Aldrich (St. Louis, MO, USA). The penicillin‐streptomycin‐neomycin, and Dulbecco's‐modified Eagle's F‐12 medium (DMEM/F12) were acquired from GIBCO BRL/Invitrogen. Antibodies specific for MMP9, MMP2, uPAR, uPA, TIMP1, TIMP2, caspase‐3, PAI‐1, NF‐κB (p65), phos‐IKK, VEGF, PARP, p‐AKT, AKT, p‐PI3K, PI3K, lamin‐B1, β‐actin and IKK were bought from Thermo Scientific. F‐actin (Alexa Fluor 488 Phalloidin) was purchased from Thermo Scientific.

Total protein was extracted from cells or tissues using a whole protein extraction kit (AS1012, ASPEN). The primary antibodies (ASPEN) used were netrin-1 (1:1000), CD63 (1:2000), CD9 (1:2000), TSG101 (1:1000), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) (1:1000), apoptosis-associated speck-like protein (ASC) (1:1000), pro-caspase-1 (1:2000), cleaved caspase-1 (1:2000), pro-IL-1β (1:2000), IL-1β (1:2000) (Abcam), Unc5b (1:2000), PI3K (1:2000), p-PI3K (1:2000), Akt (1:2000), p-Akt (1:2000), mTOR (1:2000), p-mTOR (1:2000) and GAPDH (1:2000) (26,616, Thermo). A gel image processing system (gel Pro analyser software) was used to analyse the grey value of the target strip.

Samples (≈20 μg) of identical protein concentrations were exposed to electrophoreses with 10% sodium dodecyl sulfate/polyacrylamide gel (SDS/PAGE) afterwards they were electro-transported to polyvinylidene difluoride membranes. Later, we blocked these membranes with (w/v) skimmed milk powder (5%) in PBS/Tween-20 at room temperature along two-h period. Afterwards, we incubate these membranes with polyclonal antibodies (1:1000): p-Akt (Thr450) was obtained from [Thermo Fisher Scientific, catalog number # PA5-37,469, RRID AB_2554078], p-PI3K (Tyr607) from [Thermo Fisher Scientific, catalog number # PA5-38,905, RRID AB_2555497] and p-FoxO1 (Ser249) attained by [Thermo Fisher Scientific, catalog number # PA5-64,676, RRID AB_2661988]. Then they were diluted in tris-buffered saline-tween comprising 1% bovine serum albumin and β-actin as internal control (Santa Cruz Biotechnology) diluted 1:1000 in blocking buffer. Afterwards, we incubate the membranes for one hour at room temperature with the corresponding secondary antibodies. Then they were washed and consequently developed. Lastly, photos of revealed protein bands were taken on the BioMax film (Kodak). The densitometrical quantification was performed using Image J software (Bio- Rad, California, USA). The bands densities were standardized against the corresponding density of the internal control (β-actin).