Ecl western blot detection reagent

For the analysis of IGF‐1R and ICAM‐1 protein expression, the uteri from different groups were collected, and the total protein was extracted by RIPA reagent (Solarbio) by adding 1% PMSF protease inhibitors (Solarbio) according to the standard protocol. Then, the mixtures were sonicated on ice with a Sonifier cell disruptor (75%, 5 minutes). After incubation for 30 min on ice, the mixtures were centrifuged at 16 099.2 g for 10 min at 4°C. The concentration of the supernatant was determined with a BCA protein assay kit. Ten micrograms of protein was separated by 10% or 8% SDS‐polyacrylamide gel, and then, the protein in the gel was transferred to the activated PVDF membrane. After sealing with 5% skim milk, the PVDF membranes were incubated with the corresponding IGF‐1R antibody (1:1000) (Abcam), ICAM‐1 antibody (1:1000) (Abcam) or β‐actin antibody (1:500) (Zsgb Bio) at 4°C overnight, according to the molecular weights of the different proteins. The next day, the membranes were washed with TBST three times and then incubated with anti‐rabbit IgG/HRP (1:2000) (Zsgb Bio) and goat antimouse IgG/HRP (1:2000) (Zsgb Bio) for 2 hours. Protein bands were visualized using electrochemiluminescence (ECL) Western blot detection reagents (Beyotime) under a ChemiDoc™ XRS (Bio‐Rad) system.

The same extracted proteins discussed above in section Protein Extraction and Trypsin Digestion were used in this experiment. Western blotting samples containing equal amounts of protein (20 μg/condition) were prepared by heating in 12% polyacrylamide buffer for 10 min at 95°C. Gel electrophoresis was then performed initially for 30 min at 80 V, then at 120 V until the bromophenol blue had run off the front of the gel. Following transfer to PVDF membranes (Millipore, Bedford, MA, USA) for 2 h at 80 V and 4°C, the membranes were blocked in phosphate-buffered saline (PBS) containing 5% non-fat milk and 0.1% tween-20 for 1 h at RT. Antibody incubation was performed by rinsing with TBST for 10 min three times, incubating at 4°C overnight with primary antibody (anti-acetyllysine antibody, PTM-101) diluted 1:1,000 in PBS with 1% non-fat powdered milk and 0.1% tween-20, rinsing with TBST for 10 min three times, incubating for 1 h at RT with secondary antibody (Thermo, Pierce, goat anti-mouse IgG, H+L, peroxidase-conjugated, 31430) diluted 1:5,000 in PBS with 1% non-fat powdered milk and 0.1% Tween-20), rinsing with TBST for 10 min three times, and staining with ECL western blot detection reagent (Beyotime, Beijing, China) followed by quantification using NIH Image 1.63 software (Syngene, Cambridge, UK).