Rabbit anti hif 1α

House dust mites (HDM, ALK-Abello A/S, Denmar), Recombinant Human long-isoform TSLP (lTSLP) was obtained from R&D systems. Synthetic sfTSLP peptides (63aa: MFAMKTKAALAI WCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ) were prepared by China Peptides (Shanghai, China). 3-PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) and BAY 87-2243 (Selleck, China). Rabbit anti-HIF-1α, anti-LDHA, anti-PHD and anti-VHL (proteintech, China), Rabbit anti-STAT5, anti-p-STAT5, anti-JAK1, anti-p-JAK1, anti-JAK2and anti-p-JAK2 (Cell Signaling Technology, USA), Rabbit anti-TSLPR and mouse anti-IL-7R (santa curz, USA). Rabbit anti-TSLP (Abcam, USA).

The cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested on ice. The protein concentration of the resulting lysates was determined using a RCDC protein assay kit (Sigma, USA). A prestained standard protein molecular weight marker and the protein samples were loaded into wells and separated using 8% SDS–PAGE. The blots were then transferred using electroblotting to polyvinylidene difluoride (PVDF) membranes. After the membranes were blocked with 5% nonfat dried milk or bovine serum albumin (BSA), the desired bands were incubated overnight at 4 °C with the corresponding primary antibodies, including rabbit anti-activated Notch1 (1:1000 dilution; Abcam, UK), rabbit anti-integrin β1 (1:1000 dilution; Abcam, UK), rabbit anti-HIF-1α (1:1000 dilution; Proteintech, USA) and mouse anti-β actin (1:1000 dilution; Santa Cruz, USA). Horseradish peroxidase-conjugated secondary IgG (1:5000 dilution; Proteintech, USA) was subsequently used as the secondary antibody. The results were analyzed using a ChemiDoc imaging system (Bio-Rad, USA).