Goat anti rat igg

A protein extraction kit (Beyotime, Jiangsu, PR China) was used to extract proteins from hepatic cells and liver tissue. The bicinchoninic acid method (Beyotime) was used to estimate the total protein concentration. Western blotting was then carried out according to a previously published method [17 (link)]. In this study, the antibodies used were: anti-PINK1 (catalog no. 23274-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-Parkin (catalog no. 14060-1-AP, 1:1000, Proteintech), anti-protein kinase B beta (AKT2) (catalog no. 17609-1-AP, 1:1000, Proteintech), anti-phospho-AKT2 (pAKT2) (Ser473) (catalog no. 28731-1-AP, 1:1000, Proteintech), anti-insulin receptor (INSR) (catalog no. 20433-1-AP, 1:1000, Proteintech), anti-glucose transporter type 2 (GLUT2) (catalog no. 20436-1-AP, 1:1000, Proteintech), anti-insulin receptor substrate 2 (IRS2) (catalog no. 20702-1-1AP, 1:1000, Proteintech), and anti-β-actin (catalog no. 66009-1-Ig, 1:1000, Proteintech). The secondary antibodies comprised goat anti-rabbit IgG (catalog no. SA00001-2, 1:1000, Proteintech) or goat anti-rat IgG (catalog no. GB23302, 1:1000, Servicebio, Wuhan, PR China) conjugated to horseradish peroxidase (HRP). Immunoreactive bands were captured and analyzed using Image J software (NIH, Bethesda, MD, USA).

Cells were fixed in fresh paraformaldehyde solution, and then incubated with PKM2 antibody (1:100, CST) and Cytokeratin antibody (1:100, CST) over night at 4°C and second antibodies were then loaded for 2 h at room temperature in the dark. After washing, Hoechst was used for nuclear staining. Stained cells were visualized with an Olympus Microscope IX71 (Olympus, Tokyo, Japan). Tissues from six mice per group were used for immunofluorescence staining.
Freshly isolated mouse skin specimens were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Nonspecific binding was blocked by treatment with normal goat serum and 0.3% Triton X-100. The tissues were next incubated for 24 h at 4°C with primary rat monoclonal antibodies against PKM2 (1:100, CST) and mouse polyclonal antibodies against Keratin 14 (1:100, Servicebio, Wuhan, China) diluted in primary antibody dilution buffer containing 0.3% Triton X-100. After washing with PBS, immunoreactivity was detected by incubation with FITC with goat anti-rabbit IgG (Servicebio, Wuhan, China) and Cy3 with goat anti-rat IgG (Servicebio, Wuhan, China) diluted in PBS containing 0.5% Triton X-100 for 2 h. An Olympus Microscope IX71 (Olympus, Tokyo, Japan) was used to examine the specimens.