Mtor antibody

Protease inhibitors and a lysis buffer were added to the treated cells, and the supernatant was removed after centrifugation at a low temperature to prepare the protein samples. The proteins were separated in 12% SDS-PAGE protein gel at 60 V for 30 min, separated at 100 V for 90 min for electrophoresis, and then transferred to the PVDF membrane in an ice bath (Thermal Fisher Science). After blocking with 5% skim milk for 1 h at room temperature, the membrane was incubated with the antibody overnight at 4 °C, washed 5 times for 5 min each with PBS containing 0.05% Tween-20 on a shaker. Then, the second antibody was added to the PVDF membrane and incubated at 37 °C for 1 h, followed by further washing as described above. Subsequently, the images were developed using a Western blot developer and analyzed using Image J software. The antibodies used in this article were as follows: the PRRSV nucleocapsid protein antibody (GeneTex, Irvine, CA, USA), AKT antibody (GeneTex, USA), the Phospho-AKT antibody (Ser473), the GPER1 antibody (GeneTex, USA), the mTOR antibody (Affinity, USA), the Phospho-mTOR (Ser2481) antibody (Affinity, USA), and the Actin antibody (Affinity, USA), as well as goat anti-mouse lgG and goat anti-rabbit lgG (Abcam, Cambridge, UK).

Specific pathogen-free (SPF) grade 4-week-old (70-80 g) male SD rats were purchased from Hunan Changsha Tianqin Biotechnology Co., Ltd. Common feed, high-fat feed, and streptozotocin (STZ) were all obtained from Beijing Boai Port Biotechnology Co., Ltd. Cx43 antibody (#AF0137), mTOR antibody (#AF6308), AKT antibody (#AF6261), GAPDH antibody (#T0004), β-actin antibody (#AF7018), LC3 antibody (#AF5402), p62 antibody (#AF5384), goat anti-rabbit IgG (#S0001), and goat anti-mouse IgG (#S0002) were all purchased from Affinity Biosciences (Affinity Biosciences Milwaukee, WI 53224, America). Dapagliflozin was provided by AstraZeneca Pharmaceutical Co., Ltd.