Quantitative Immunoblotting of Signaling Pathways

Immunoblotting of lysates from cells and frozen tumor fragments was performed as described [52 (link)]. Immunoprecipitation of p85 from cell lysates was performed using Dynal protein-G beads (Invitrogen) and p85 antibody (Abcam) as described [53 ]. Following SDS-PAGE, proteins were transferred to nitrocellulose membranes, and even protein loading was visually confirmed by Ponceau S staining. Blots were probed with antibodies against P-AKT_S473, P-AKT_T308, AKT, P-p70S6K_T389, P-S6_S240/244, P-IGF-1Rβ_Y1135/6/P-InsRβ_Y1150/1, IGF-1Rβ, InsRβ, IRS-1, IRS-2, actin, vinculin (Cell Signaling), p85 (Abcam), or ER (Santa Cruz). HRP-labeled secondary antibodies (GE Healthcare) and ECL substrate (Pierce) were used for signal detection. Densitometry analysis of immunoblot film was performed using ImageJ software, and relative signal values were analyzed by ANOVA followed by Bonferroni multiple comparison-adjusted post-hoc test between groups.

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Yang W., Schwartz G.N., Marotti J.D., Chen V., Traphagen N.A., Gui J, & Miller T.W. (2018). Estrogen receptor alpha drives mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer. Oncotarget, 9(10), 8810-8822.

Publication 2018

Dynal protein-G beads vinculin HRP-labeled secondary antibodies

Actin Anova Antibodies Antibody Cell Densitometry Frozen Igf 1 Immunoblot analysis Immunoprecipitation Irs 1 Irs 2 Membranes Nitrocellulose Ponceau s Protein Protein g Sds page Signal detection Tumor Vinculin

Corresponding Organization : Dartmouth–Hitchcock Medical Center

Other organizations : Dartmouth College

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Variable analysis

independent variables

Cell lysates and frozen tumor fragments

dependent variables

Phosphorylation levels of AKT (S473 and T308), p70S6K (T389), S6 (S240/244), IGF-1Rβ (Y1135/6)/InsRβ (Y1150/1)
Protein levels of AKT, IGF-1Rβ, InsRβ, IRS-1, IRS-2, actin, vinculin, p85, ER

control variables

Protein loading confirmed by Ponceau S staining

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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