Western Blot Analysis of Signaling Proteins

Cells or tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors (Roche Diagnostics, IN). Thirty micrograms of protein resolved by SDS-PAGE was immunoblotted as previously described (15 (link)). Membranes were probed with the following antibodies: α-tubulin (2144), β-actin (47778), AMPK (2603), p-AMPK (T172; 2535), HSF1 (4356), p-AKT (S473; 4060), p-AKT (T308; 2965), AKT (9272), p-mTOR (S2448; 2971), mTOR (2972), p-p70SK (T389; 9206), p70SK (9202), p-S6R (S240/244; 5364), S6R (2217), p-4E-BP1 (T37/46; 2855), p-CAD (S1859; 12662), p-GSK3 (S9/21; 9336), 4E-BP1(S65; 9451), Pten (9559), p-PDK1 (S241; 3438), p-ERK (4370), 4E-BP1(T70; 9455), and 4E-BP1 (9452) (Cell Signaling Technology), α-tubulin (5286), HSF1 (13516) and ERK (sc-94) (Santa Cruz Biotech) and c-Myc (790–4628) (Hoffmann La Roche).

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Eroglu B., Pang J., Jin X., Xi C., Moskophidis D, & Mivechi N.F. (2019). HSF1-mediated Control of Cellular Energy Metabolism and mTORC1 Activation Drive Acute T Cell Lymphoblastic Leukemia Progression. Molecular cancer research : MCR, 18(3), 463-476.

Publication 2019

protease and phosphatase inhibitors α-tubulin p-AMPK p-AKT p-4E-BP1 4E-BP1 p-PDK1 p-ERK

4e bp1 Actin Antibodies Buffer C myc Cells Diagnostics Gsk3 Inhibitors Membranes Mtor Pdk1 Phosphatase Protease Protein Pten Ripa Sds page Tissues α tubulin

Corresponding Organization : Augusta University

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Variable analysis

independent variables

Cells or tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors (Roche Diagnostics, IN)

dependent variables

Protein expression and phosphorylation levels of the following proteins: α-tubulin, β-actin, AMPK, p-AMPK (T172), HSF1, p-AKT (S473), p-AKT (T308), AKT, p-mTOR (S2448), mTOR, p-p70SK (T389), p70SK, p-S6R (S240/244), S6R, p-4E-BP1 (T37/46), p-CAD (S1859), p-GSK3 (S9/21), 4E-BP1 (S65), Pten, p-PDK1 (S241), p-ERK, 4E-BP1 (T70), and 4E-BP1

control variables

30 micrograms of protein resolved by SDS-PAGE

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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