Anti cdk4

Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone (HyClone, Logan, CT, USA). Fetal bovine serum (FBS) was purchased from ABGENT (San Diego, CA, USA). We purchased penicillin, streptomycin, and Angiotensin II from Sigma-Aldrich (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Kumamoto, Japan). The rat recombinant Klotho protein was from Cloud-Clone Corporation (Houston, TX, USA). The antibodies used in this study were as follows: mouse monoclonal PCNA (PC10), rabbit polyclonal NF-κB p65 (C-20), rabbit polyclonal p-NF-κB p65 (Ser 536), rabbit polyclonal ERK1/2 (K-23), rabbit polyclonal p-ERK1/2 (Thr202/Tyr204), rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FL-335) (Santa Cruz Biotechnology, CA, USA); and rabbit polyclonal SM22α (Cloud-Clone Corp., Wuhan, China). Anti-CDK2, anti-CDK4, anti-cyclin D, and anti-cyclin E antibodies were purchased from Wanleibio, Shenyang, China, and anti-Akt and anti-phospho-Akt (Ser473) antibodies were from Cell Signaling (MA, USA). The reactivity with rat antigens is included in all of these antibodies. Through ImageJ software (NIH, USA), the signal intensity was quantified.