Rabbit anti gapdh

After 48 h of cell treatment, a protein lysis buffer was used to extract the total protein and the protein concentration was determined using a BCA kit (CoWin Biosciences, Beijing). Following the addition of the same amount of protein, polyacrylamide gel electrophoresis was implemented using 100 g/L sodium lauryl sulfate. Once the electrophoresis was completed, the protein was transferred onto a 0.45 μm PVDF membrane, which was then sealed in a confining liquid. The phosphorylated and nonphosphorylated proteins were sealed using 5% fetal bovine serum and 50 g/L skimmed milk powder, respectively. After sealing, the primary and secondary antibodies were added accordingly. This point represents the completion of the method development. The primary antibodies used here were as follows: rabbit anti‐GAPDH (1:5000, Affinity, USA); rabbit anti‐Nur77 (1:1000, Proteintech, USA); and rabbit anti‐CXCR4, rabbit anti‐PI3K and p‐PI3K, AKT, and p‐AKT (all 1:1000, Abcam, UK). The secondary antibody used here was goat anti‐rabbit IgG (1:1000, CST, USA). Each experiment was repeated in triplicate.

The collected protein samples were quantified using BCA method (Yeasen, China) and separated by 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane (Millipore, Germany). After blocking with 5% non-fat milk at room temperature for 1 h, the membrane was incubated with primary antibodies including rabbit anti-PER1 (Affinity, USA, 1:800), rabbit anti-MMP13 (Proteintech, China, 1:1000), rabbit anti-NF-kB p65 (Affinity, USA, 1:500), rabbit anti-Phospho-NF-kB p65 (Ser536) (Affinity, USA, 1:500) and rabbit anti-GAPDH (Affinity, USA, 1:2000) at 4 °C for 16 h. Following the washing with 0.1% TBST, the secondary antibody (Beyotime, China) was incubated with the membrane. After washing with 0.1% TBST, a chemiluminescence ECL system (Millipore, Germany) was used to visualize the immunoreactive proteins.

The collected protein samples were quantified using BCA method (Yeasen, China) and separated by 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane (Millipore, Germany). After blocking with 5% non-fat milk at room temperature for 1 h, the membrane was incubated with primary antibodies including rabbit anti-PER1 (Affinity, USA, 1:800), rabbit anti-MMP13 (Proteintech, China, 1:1000), rabbit anti-NF-kB p65 (Affinity, USA, 1:500), rabbit anti-Phospho-NF-kB p65 (Ser536) (Affinity, USA, 1:500) and rabbit anti-GAPDH (Affinity, USA, 1:2000) at 4 °C for 16 h. Following the washing with 0.1% TBST, the secondary antibody (Beyotime, China) was incubated with the membrane. After washing with 0.1% TBST, a chemiluminescence ECL system (Millipore, Germany) was used to visualize the immunoreactive proteins.