Protein Extraction and Western Blot

Tissue was homogenized, or cultured cells washed, and then lysed in complete RIPA buffer as described previously [50 (link)]. Primary antibodies (Ab) used: AR (sc-816), PI3K p110β (sc-376641), β-actin (sc-47778), GAPDH (sc-32233), and α-tubulin (sc-5286) from Santa Cruz Biotechnology (Santa Cruz, CA); AKT1 (#2938), AKT2 (#3063), AKT3 (#8018), AKT3 (#14982), AKT^poS473 (#9018), AKT^poS473/4 (#9271), AKT2^poS474 (#8599), AKT^poT308 (#4056S), phospho-AKT substrate RXXS*/T* (#9614S), PRAS40 (#2691S), PRAS40^poT46 (#2997T), PTEN (#9552), and PI3K p110α (#4249) from Cell Signaling Technologies (Beverly, MA); SMAD4 (ab-40759) from Abcam (Cambridge, MA); SSeCKS/Akap12 [12 (link)]. Between 15 and 35 µg of total protein per sample was separated by SDS-PAGE for IB.

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Miller K.A., Degan S., Wang Y., Cohen J., Ku S.Y., Goodrich D.W, & Gelman I.H. (2023). PTEN-regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression. Oncogene, 43(1), 22-34.

Publication 2023

β-actin (sc-47778), GAPDH (sc-32233), α-tubulin (sc-5286) AKTpoS473 AKTpoS473/4 AKT2poS474 AKTpoT308 PRAS40 PI3K p110α SMAD4 (ab-40759)

Corresponding Organization : Cancer Genetics (United States)

Other organizations : American Society of Human Genetics, Harvard University, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Tissue homogenization/cell culture
Lysis in complete RIPA buffer

dependent variables

Protein expression/abundance of AR, PI3K p110β, β-actin, GAPDH, α-tubulin, AKT1, AKT2, AKT3, phospho-AKT (S473, S473/T474, S474, T308), phospho-AKT substrate (RXXS*/T*), PRAS40, phospho-PRAS40 (T46), PTEN, PI3K p110α, SMAD4, SSeCKS/Akap12

control variables

Amount of total protein per sample (15-35 µg)

controls

Positive controls: None specified
Negative controls: None specified

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