Anti akt1 akt2 akt3

Sheep (ALC and MF) and mouse skin tissues were fixed in 4% paraformaldehyde formalin in PBS at 4 °C overnight, embedded in paraffin, sectioned at 5 μm, and stained with a hematoxylin and eosin staining kit (H&E). The following antibodies were used for immunostaining: anti-Ki67 (Abcam, ab15580), anti-Caspase 3 (Abcam, ab184787), anti-AKT1 + AKT2 + AKT3 (Abcam, ab179463); phospho-PI3-kinase p85-alpha/gamma (Abmart, T40116F); mTOR antibody (Abmart, T55306); HRP-labeled goat anti-rabbit IgG(H+L) (Beyotime, A0208), and HRP-labeled goat anti-mouse IgG(H+L) (Beyotime, A0216). The signal was detected using the DAB Horseradish Peroxidase Color Development Kit (Beyotime, P0202), and the sections were stained with hematoxylin. Photographs were taken using a microscope (ECHO, RVL-100-G, USA).

The proteins of mouse skin, ALC and MF wool lamb skin, and sFFCs (transfected with miRNA mimics, NC, inhibitor, sITS-miRNA mimics, and sITS-miRNA NC) were extracted and electrophoresed in a 10% SDS/polyacrylamide gel (SDS-PAGE Gel Quick Preparation K) and transferred to a 0.45 μm Polyvinylidene fluoride, PVDF membrane. The blots were blocked in QuickBlock™ Blocking Buffer for western blot (Beyotime, Shanghai, China) for 1 h and probed with antibodies (for mouse skin western blot) phospho-PI3-kinase p85-alpha/gamma (Abmart, T40116F, China); anti-AKT1 + AKT2 + AKT3 (Abcam, ab179463, USA); mTOR antibody (Abmart, T55306, China); and β-actin antibody (Beyotime, AA128, China) in primary antibody dilution buffer (Beyotime, P0023A, China) overnight. The antibodies for sheep skin and sFFCs were: phospho-PI3-kinase p85-alpha (Tyr607) antibody (Affinity, AF3241, USA); anti-AKT (GeneTex, GTX128415, USA); and mTOR antibody (GeneTex, GTX101557, USA). Signals were detected using HRP-labeled secondary antibodies and a chemiluminescent substrate (BeyoECL Moon) (Beyotime, A0208 or A0216, China).