Details about WB analysis conditions are reported in Supplementary Table 1 . In brief, after cell lysis, equal amount of proteins was loaded on a 4–20% gradient gel (BioRad) and separated by SDS-PAGE. The membrane was further probed with antibodies against CDK4 (EPR4513-32-7, abcam, dilution 1:1000) and CDKN2A (G175-405, BD Pharmingen, San Jose, CA, USA, dilution 1:500).
It is also known that CDK4, along with CDK6, activated by binding to D-type cyclins, is responsible for the G1-S phase transition in the cell cycle by phosphorylating and inhibiting the retinoblastoma (RB) protein and the related proteins p107/RBL1 and p130/RBL2 (36 (link)). Moreover, CDK4/6 inhibitors have been reported to reduce cell viability in ACC cell lines despite lack of pRb expression (28 (link), 31 (link)) p130/RBL2 being already reported to be expressed in the NCI-H295R cell line (28 (link)). Thus, we investigated RB and p130/RBL2 protein expression in both cell lines. Signal detection was achieved by incubation with appropriate HRP-labeled secondary antibodies and Amersham ECL Prime reagent visualizing the protein-antibody complex by enhanced chemiluminescence.
It is also known that CDK4, along with CDK6, activated by binding to D-type cyclins, is responsible for the G1-S phase transition in the cell cycle by phosphorylating and inhibiting the retinoblastoma (RB) protein and the related proteins p107/RBL1 and p130/RBL2 (36 (link)). Moreover, CDK4/6 inhibitors have been reported to reduce cell viability in ACC cell lines despite lack of pRb expression (28 (link), 31 (link)) p130/RBL2 being already reported to be expressed in the NCI-H295R cell line (28 (link)). Thus, we investigated RB and p130/RBL2 protein expression in both cell lines. Signal detection was achieved by incubation with appropriate HRP-labeled secondary antibodies and Amersham ECL Prime reagent visualizing the protein-antibody complex by enhanced chemiluminescence.
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