A17909

The ultrasonic cell crusher (SM-650A, SHUNMATECH) was used for submandibular gland tissue protein extraction, the prepared minced tissue was placed in it for cell disruption and then centrifuged for cell debris, and the whole protein was extracted. The experiment begun with SDS-PAGE electrophoresis for separating proteins of different molecular weights. Then, proteins were transferred to polyvinylidene fluoride (PVDF) membranes, washed with TBST for 1 min, and blocked with phosphate-buffered saline (PBS) containing 5% blotting grade for 1 h. Followed by blocking, three times washing each for 5 min was performed with Tris-buffered saline with Tween 20 (TBST). The appropriate primary antibody (1 : 1000) incubation was performed on transferred membrane at 4°C overnight and then followed by 1 h IgG (1 : 2000, ABclonal) incubation at room temperature. The protein band imaging was performed by the enhanced chemiluminescence (ECL) system following the manufacturer's instructions. The catalogue number and brand of used primary antibodies were listed in the following: anti-PI3K (4228T, ABclonal), anti-p-PI3K (4228T, CST), anti-AKT (A17909, ABclonal), anti-p-AKT (4060T, ABclonal), anti-HIF-1α (A16873, ABclonal), anti-VEGF (A12303, ABclonal), anti-IL-17A (A0688, ABclonal), anti-LC3 I/II (A19665, ABclonal), and β-actin (1 : 2000, AC026, ABclonal).

Western blotting assays were conducted as previously reported [18 (link)]. The primary antibodies were as follows PR55α (1/1000, A2185, Abclonal, China), AKT (1/1000, A17909, Abclonal, China), p-AKT-T308 (1/1000, AP0304, Abclonal, China), P-AKT-S473 (1/1000, AP0140, Abclonal, China), ERK1/2 (1/1000, A4782, Abclonal, China) and p-ERK1/2-T202/Y204 (1/1000, AP0472, Abclonal, China).