Western Blot Analysis of Cell Signaling

Cell pellets were lysed in 2x Laemmli buffer (Alfa Aesar) and performed as described previously^{12 (link)} with the following antibodies: anti-PTEN (A2B1, Santa Cruz Biotechnology, sc-7974, 1:1000), anti-GAPDH (V-18, Santa Cruz Biotechnology, sc-20357, 1:500), anti-P65 (L8F6, Cell Signaling Technology, 6956, 1:1000), and anti-phospho-P65 Ser536 (93H1, Cell Signaling Technology, 3033, 1:1000). As secondary antibodies anti-mouse IRDye 800 (1:5000; Li-Cor Biosciences, Cat# 926-68072), anti-rabbit IRDye 680 (1:5000; Li-Cor Biosciences, Cat# 926–6807), and anti-goat IRDye 680 (1:5000; Li-Cor Biosciences, Cat# 926-32224) were used. The blot was analyzed by Odyssey CLx imaging system (Li-Cor).

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Kwon B., Fansler M.M., Patel N.D., Lee J., Ma W, & Mayr C. (2022). Enhancers regulate 3′ end processing activity to control expression of alternative 3′UTR isoforms. Nature Communications, 13, 2709.

Publication 2022

2x Laemmli buffer anti-PTEN (A2B1, anti-GAPDH (V-18, anti-phospho-P65 Ser536 (93H1, anti-mouse IRDye 800 anti-rabbit IRDye 680 Odyssey CLx imaging system

Antibodies Cell Gapdh Goat Irdye 800 Laemmli buffer Mouse Pellets Pten Rabbit

Corresponding Organization :

Other organizations : Memorial Sloan Kettering Cancer Center, Tri-Institutional PhD Program in Chemical Biology, Cornell University

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Variable analysis

independent variables

Cell pellets were lysed in 2x Laemmli buffer (Alfa Aesar)

dependent variables

Levels of PTEN, GAPDH, P65, and phospho-P65 Ser536 proteins

control variables

Antibodies used: anti-PTEN (A2B1, Santa Cruz Biotechnology, sc-7974, 1:1000), anti-GAPDH (V-18, Santa Cruz Biotechnology, sc-20357, 1:500), anti-P65 (L8F6, Cell Signaling Technology, 6956, 1:1000), and anti-phospho-P65 Ser536 (93H1, Cell Signaling Technology, 3033, 1:1000)
Secondary antibodies used: anti-mouse IRDye 800 (1:5000; Li-Cor Biosciences, Cat# 926-68072), anti-rabbit IRDye 680 (1:5000; Li-Cor Biosciences, Cat# 926–6807), and anti-goat IRDye 680 (1:5000; Li-Cor Biosciences, Cat# 926-32224)
Blot analysis using Odyssey CLx imaging system (Li-Cor)

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