Rabbit anti alix

The contralateral side of the uterus (n = 6 per group) was homogenised in RIPA lysis solution (Solarbio) containing 1 mM PMSF. The concentration of the extract was determined by centrifuging it at 12,000 rpm for 5 min and then analysing the supernatant using a BCA protein assay kit. Electrophoretically, a 4–20% polyacrylamide gradient gel was used to separate the 30 g of extracted protein. Milk at 5% in TBST was used to inhibit nonspecific binding for 1 h. Primary antibodies were used to detect the following proteins: rabbit anti-Alix, rabbit anti-CD63, mouse anti-GAPDH, rabbit anti-VEGF (1:1000; Santa Cruz), mouse anti-LIF, rabbit anti--Tubulin (1:1000; Antibody Revolution), and mouse antiintegrin-3. After that, the membranes were treated with a fluorescently labelled IRDye 800 secondary antibody (1:3000; Rockland) of the matching species for 2 h at room temperature in the dark. The LICOR Odyssey (Lincoln, NE) was used to take digital pictures of the protein bands. ImageJ software (NIH, Bethesda, MD, USA) was used to measure the grey value of the protein band to determine the relative expression. The levels of -Tubulin were utilised as a reference. At least three separate trials were conducted.