Ab76003

Total proteins from cells and tissues were extracted by the RIPA lysate. Protein quantification was conducted with a BCA protein quantification kit. An appropriate amount of protein sample was denatured at 100°C for 3–5 min and then subjected to protein electrophoresis using 10% SDS-PAGE for 1 h. the samples were transferred to PVDF membranes. The membranes were blocked in Tris Buffered Saline with Tween 20 blocking solution containing 5% skim milk powder at room temperature for 2 h. The membranes were washed three times with TBST and incubated overnight at 4°C using primary antibodies against MMP-2, MMP-9, p-PI3K, PI3K, p-AKT, AKT, and GAPDH (ab92536, ab76003, ab278545, ab140307, ab192623, ab8805, ab8245; all from Abcam, Shanghai, China). The next day, the horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG (ab96899) and goat anti-mouse secondary antibody IgG (ab96879) dilution buffer were added, and membranes were incubated with these secondary antibodies at room temperature for 2 h. The enhanced chemiluminescence reagent was added dropwise, and the membranes were imaged by the gel imaging system. The gray value of each band was analyzed by the ImageJ software. The results were expressed as the ratio of the gray value of the target protein to GAPDH, the internal reference protein.

For immunohistochemical analyses, the sections were exposed overnight at 4°C to antibodies against E-cadherin (rabbit monoclonal; 1 : 200; ab76319; Abcam), COL I (rabbit monoclonal; 1 : 200; ab270993; Abcam, Cambridge, UK), MMP-9 (rabbit monoclonal; 1 : 200; ab76003; Abcam), TIMP-1 (rabbit polyclonal; 1 : 200; Abcam), α-SMA (mouse monoclonal; 1 : 200; ab7817; Abcam), or fibronectin (rabbit monoclonal; 1 : 200; ab199056; Abcam), followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1 : 500; 115-035-003; Jackson ImmunoResearch, Ely, UK).

PBS was used to wash fresh tissue which was grounded into a homogenate, and then RIPA buffer was used to lyse them to obtain total protein. Protein concentrations were determined by the BCA assay (Solarbio, PC0020, China). Samples (30 μg protein) were resolved on 8% SDS-PAGE gel and transferred to PVDF at 4°C. Membranes were blocked in fat-free milk combined with TBST for 1 hour at 25°C and immunoblotted with the appropriate diluted primary antibody KIF20A (Abcam, ab104118, UK; 1 : 1000 dilution for Western blot and 1 : 200 dilution for IHC-P), PCNA (Abcam, ab92552, UK; 1 : 1000 dilution for Western blot), Ki67 (Abcam, ab16667, UK; 1 : 1000 dilution for Western blot), MMP2 (Abcam, ab37150, UK; 1 : 500 dilution for Western blot), MMP9 (Abcam, ab76003, UK; 1 : 5000 dilution for Western blot), and GAPDH (SunGene Biotech, KM9002, China; 1 : 5000 dilution for Western blot) at 4°C overnight. Then, membranes were washed 10 minutes three times and incubated with the secondary antibody for 1 hour at 25°C. Finally, the blots were visualized by Imager.

Implant neutrophil and macrophage numbers were determined by staining for Myeloperoxidase (MPO; ab90810, Abcam) and CD68 (ab31630, Abcam) respectively. Cellular localisation of NGAL and MMP-9 in implant inflammatory cells and isolated peripheral blood neutrophils was determined using antiNGAL (M145, SantaCruz) and antiMMP-9 (sc-6840, SantaCruz) antibodies according to standard procedures [19 (link)]. The secondary antibodies (Alexa Fluor 488 or 594, Invitrogen) were used for visualisation and the nuclei were stained with DAPI. Images were captured using an Olympus AX-70-Fluorescence microscope.
The cellular localisation of NGAL (ab41105; Abcam) and MMP-9 (ab76003; Abcam) was examined in skin wound tissue by immunohistochemistry. For quantitation of NGAL, the staining intensity was examined in the epithelial (top), granulation tissue (middle) and inflammatory (basal) zones and scored by two independent observers based on a scale of 0 = no staining above isotype control to 3 = intense staining. The score for each zone was summed and the data was analysed using Chi squared analysis at an intensity cut off at ≥ 6. MMP-9 staining was localised mainly to cells in the granulation tissue layer and the staining intensity in this area was quantified using Image J software.

The following primary antibodies were used: antibodies against Rictor (ab70374), MMP‐2 (ab37150) and MMP‐9 (ab76003) from Abcam (Cambridge, USA); antibodies against phospho‐AKT (S473) (#9721) and phospho‐AKT (T308) (#13038) from Cell Signaling Technology; and antibodies against AKT (AF6259), phospho‐CDK2 (Thr160) (AF3237), phospho‐Histone H3.1 (Ser10) (AF3358) and β‐actin (T0022) from Affinity Biosciences. HRP‐conjugated goat anti‐rabbit IgG and anti‐mouse IgG secondary antibodies were obtained from Santa Cruz (Dallas, TX, USA). Gelatin (G7041) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). MK‐2206, 8‐[4‐(1‐aminocyclobutyl)phenyl]‐9‐phenyl‐1,2,4‐triazolo [3,4‐f] 1, 6naphthyridin‐3(2H)‐one hydrochloride [1:1], was obtained from Selleck (Shanghai, China). 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) was purchased from Sigma‐Aldrich (St. Louis, MO).