Hrp conjugated goat anti rabbit igg mab

Cells were lysed by RIPA lysate (plus protease inhibitor) (Beijing ComWin Biotech Co., Ltd., Beijing, China) to extract the total protein and protein concentration was determined by a BCA method. Cell lysates dissolved in SDS buffer were segregated using 10% SDS-PAGE (20 µg protein were loaded for each lane) and electro-transferred to a PVDF membrane. The membrane was blocked by 5% skimmed milk for 1 h and probed with specific primary antibodies [PABPC1L (dilution 1:1,000; cat. no. ab233280), PI3K (dilution 1:1,000; cat. no. ab232997) and p-PI3K (dilution 1:1,000, cat. no. ab182651) (all from Abcam, Cambridge, MA, USA), and AKT (dilution 1:1,000; cat. no. 4691), p-AKT (Ser473) (dilution 1:1,000; cat. no. 4060) and GAPDH (dilution 1:5,000; cat. no. 5174) (all from Cell Signaling Technology, Inc., Danvers, MA, USA)] overnight at 4°C. Then, the membrane was incubated with the HRP-conjugated goat anti-rabbit IgG mAb (dilution 1:5,000; PV-6001; ZSGB-BIO, Beijing, China) at room temperature for 1 h. Then, the signals were detected using an Immobilon™ western chemiluminescent HRP substrate (Millipore) and analyzed by a gel imaging analysis system (Bio-Rad Laboratories, Inc.). Detailed procedures were described previously (14 (link)).

HMEC-1 cells and lung endothelial tissue from experimental rats were fixed using 10% formaldehyde for 2 h at 37°C and then embedded in paraffin. For HMEC-1 cells, rat anti-human SEMA-7A antibody (1:1,000, cat no. ab23578; Abcam) labeled with fluorescein isothiocyanate (green fluorescence) for 12 h at 4°C was used to analyze SEMA-7A expression prior to and following treatment with Anti-SEMA-7A (2 mg/ml), SEMA-7A (2 mg/ml) or PBS (2 mg/ml) for 2 h at 4°C. Lung tissues were cut into sections 4-µm thick. Antigen retrieval was performed in tissue sections and the sections were incubated with the following primary antibodies: SEMA-7A (1: 1,000; cat no. ab23578), M1-7 (1:1,000; cat no. ab61108) and FSF/FKF (1:1,000; cat no. ab200478; all Abcam) for 8 h at 4°C and correlative secondary antibodies: HRP-conjugated goat anti-rabbit IgG mAb (1:2,000; cat no. PV-6001; ZSGB-BIO, Beijing, China) were applied for specimens for 24 h at 4°C. The staining of the slides was performed with the avidin-biotin-peroxidase complex. A Ventana Benchmark automated staining system (Version 3.0, Ventana Medical Systems, Inc; Roche Diagnostics, Basel, Switzerland) was used to analyze SEMA-7A, ERF, ERK, M1-7 and FSF/FKF levels using confocal microscopy (Carl Zeiss LSM780, Carl Zeiss AG, Oberkochen, Germany).