Western Blot Protein Analysis Protocol

Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [41 (link)]. Antibodies against p-Rb^ser780, p-Rb^{ser807/ser811}, Rb, Cyclin D1, p16^INK4a, p-CDK6, CDK6, p-AKT^ser473, AKT, pEGFR^tyr1068, EGFR, p53, p-MDM2, p21, c-Myc, actin, and HRP-conjugated secondary antibodies was from Cell Signaling Technology; the chemiluminescence system (Immobilion^TM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA, USA). Reagents for electrophoresis and blotting analysis were from BIO-RAD (Hercules, CA, USA). The whole Western blots are shown in Figures S3–S5.

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La Monica S., Fumarola C., Cretella D., Bonelli M., Minari R., Cavazzoni A., Digiacomo G., Galetti M., Volta F., Mancini M., Petronini P.G., Tiseo M, & Alfieri R. (2020). Efficacy of the CDK4/6 Dual Inhibitor Abemaciclib in EGFR-Mutated NSCLC Cell Lines with Different Resistance Mechanisms to Osimertinib. Cancers, 13(1), 6.

Publication 2020

p-Rbser807/ser811 p-AKTser473 pEGFRtyr1068 p-MDM2 c-Myc ImmobilionTM Western Chemiluminescent HRP Substrate

Actin Antibodies C myc Cdk6 Chemiluminescence Cyclin d1 Egfr Electrophoresis Mdm2 P16ink4a Protein Western blots

Corresponding Organization : University of Parma

Other organizations : Istituto Nazionale per l'Assicurazione Contro gli Infortuni sul Lavoro, Institut de Recherche en Cancérologie de Montpellier

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Variable analysis

independent variables

Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE

dependent variables

Levels of p-Rb^ser780, p-Rb^ser807/ser811, Rb, Cyclin D1, p16^INK4a, p-CDK6, CDK6, p-AKT^ser473, AKT, pEGFR^tyr1068, EGFR, p53, p-MDM2, p21, c-Myc, actin

control variables

Reagents for electrophoresis and blotting analysis

controls

Positive control: None specified.
Negative control: None specified.

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