Western Blot Analysis of Protein Lysates

Protein lysates (40 μg) were prepared in the presence of protease inhibitors and analyzed as described previously (18 (link)) using 4–20% Criterion gels (Bio-Rad, Hercules, CA, USA). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (YAP1, RB1, CDKN2A/B, NEUROD1, CCND1, CDK4, CDK6, pRB-s780), Santa Cruz (Dallas, TX, USA) (INSM1, CDK2, E2F1), Sigma (St. Louis, MO, USA) (Actin, POU2F3), and BD Biosciences (Franklin Lakes, NJ, USA) (ASCL) and are listed in Sup. Table 1. Bands on western blots were quantified using Image J.

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Wildey G., Shay A.M., McColl K.S., Yoon S., Shatat M.A., Perwez A., Spainhower K.B., Kresak A.M., Lipka M., Yang M., Behtaj M., Fu P., Alahmadi A., Mneimneh W., Abbas A, & Dowlati A. (2023). Retinoblastoma expression and targeting by CDK4/6 inhibitors in small cell lung cancer. Molecular cancer therapeutics, 22(2), 264-273.

Publication 2022

4–20% Criterion gels NEUROD1 CCND1 pRB-s780 Actin

Actin Antibodies Ccnd1 Cdk2 Cdk6 Cdkn2a E2f1 Gels Neurod1 Pou2f3 Protease inhibitors Protein Western blots Yap1

Corresponding Organization : Case Western Reserve University

Other organizations : Louis Stokes Cleveland VA Medical Center

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Variable analysis

independent variables

Protein lysates

dependent variables

Protein expression levels (YAP1, RB1, CDKN2A/B, NEUROD1, CCND1, CDK4, CDK6, pRB-s780, INSM1, CDK2, E2F1, Actin, POU2F3, ASCL)

control variables

Protease inhibitors
4–20% Criterion gels (Bio-Rad, Hercules, CA, USA)
Antibodies purchased from Cell Signaling Technology (Danvers, MA, USA), Santa Cruz (Dallas, TX, USA), Sigma (St. Louis, MO, USA), and BD Biosciences (Franklin Lakes, NJ, USA)
Quantification using Image J

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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