Ab119992

The cells or exosomes were lysed with RIPA lysate (Sigma) to obtain proteins. The BCA Assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. An equal amount of protein was added onto 10% SDS-PAGE gels, and the protein was then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The blots were then incubated with primary antibodies, Anti-PI3Kγ antibody (1 : 1,000, ab32089, Abcam), Anti-CD163 antibody (1 : 1,000, ab213612, Abcam), Anti-CD206 antibody (1 : 1,000, sc-58986, Santa Cruz Biotechnology), Anti-PTEN antibody (1 : 1,000, ab267787, Abcam), Anti-AKT antibody (1 : 1,000, ab213612, Abcam), Anti-pAKT antibody (1 : 1,000, ab38449, Abcam), Anti-Vimentin antibody (1 : 1,000, ab20346, Abcam), Anti-α-SMA antibody (1 : 1,000, ab108424, Abcam), Anti-IL-10 antibody (0.5 µg/ml, ab134742, Abcam), Anti-CD63 antibody (1 µg/ml, ab38418, Abcam), Anti-CD61 antibody (1 : 1,000, ab119992, Abcam), Anti-HSP70 antibody (1 : 1,000, ab2787, Abcam), and anti-Actin (1 µg/ml, ab8286, Abcam) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was then added to incubate the blots at room temperature for 2 h. At last, the blots were visualized with the Novex™ chemiluminescent substrate reagent kit (Waltham, MA, USA).

Western blotting analysis was performed as previously described [34 (link)] using antibodies against METTL3 (1:1,500; ab221795, Abcam), HOXA10 (1:2,000; A8550, Abclonal), ITGB3 (1:1,000; ab119992, Abcam), EMX2 (1:1,500; ab171818, Abcam) and GAPDH (1:6,000; KC-5G5, Aksomics). GAPDH was used as an endogenous control to normalize protein loading. The relative band intensities were measured using a quantitative scanning densitometer and image analysis software, ImageJ.

Cells were lysed in whole cell lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Lysates were resolved on SDS-PAGE and Western blotted with antibodies for Kindlin3 (Abcam; #ab68040), Talin1 (Abcam; #ab71333) and integrinβ3/CD61 (Abcam; #ab119992).

Fibroblast cells cultures were treated with 10 μg ml⁻¹ of α5, β1, β3 integrins, or combinations of α5/β 1, αv/β3 blocking antibodies and were analysed after 24 h. The antibodies used are: β1 integrin (Millipore AB1952); β3 integrin (abcam ab119992); α5 (abcam ab72663); α5 β3 (abcam ab78289).

A protein extraction kit (Ding Guo, China) was used to extract total cell protein. The extracted proteins from platelets, Pex and PexD were boiled for 5 min and separated by SDS-PAGE. Coomassie blue staining was used to visualize proteins. Proteins were prepared for western blotting as previously stated. Following gel electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad). After 2 h of blocking in 5% skim milk, the membranes were incubated with anti-TSG101 (Abcam, ab125011), anti-CD9 (Abcam, ab92726), anti-CD63 (Abcam, ab217345), anti-CD61 (Abcam, ab119992), anti-CD41 (Abcam, ab63983) or anti-P-selectin (Abcam, ab6632). The antibodies were added at a dilution of 1:1000 and incubated at 4 °C overnight, and the blots were then incubated with 5% skim milk for 2 h. Then, the membranes and an appropriate secondary antibody (1:10,000) were incubated at room temperature for 1 h.