P jnk

Radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) was used to prepare tissue lysates with 1% SDS. Protein quantification was performed using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Total proteins were resolved on 10% SDS-PAGE and electrotransferred onto Hydrophobic PVDF Transfer Membrane (MilliporeSigma, Temecula, CA, USA). The membranes were blocked in 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min and incubated overnight at 4°C with primary rabbit polyclonal antibodies against HIF-1α (cat. no. AF1009; Affinity), VEGF (cat. no. AF5131; Affinity), DLK-1 (cat. no. AP20860c; Abgent), p-JNK (cat. no. AF3318; Affinity), JNK (cat. no. AF6318; Affinity), and GAPDH (cat. no. A01020; Abbkine). All antibodies were diluted 1:1,000 in Tris-buffered saline. Blots were washed in TBST and labeled with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA). Bands and band intensity were detected and calculated using chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) and Image Quant LAS4000 (GE Healthcare Life Sciences, Little Chalfont). The Protein expression levels were expressed related to GAPDH levels 32 (link), 33 (link).