Protein Extraction and Signaling Pathway Analysis

Proteins were extracted using 1X RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA), and concentration determined using the BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA). Immunoblots (western blot) were performed as previously described [48 (link)]. Primary antibodies against ERK1/2, p-ERK1/2 (Thr202/Tyr204), MEK1/2, p-MEK1/2 (Ser217/221), p-PKCα/β II (Thr638/641), CAMKII, p-CAMKII (Thr286), BIM; p-BIM (Ser69), CHOP, Bcl-2, Bcl-xL, p-eIF2α (Ser51), STIM1, PARP, NEDD8 and secondary HRP-conjugated antibodies were obtained from Cell Signaling (Danvers, MA). Antibodies for PKCβ, luciferase, ERO1 and Orai1 were purchased from Santa Cruz Biotechnology (Dallas, TX). Proteins expression was determined by densitometry analysis of the immune-detected bands, normalized to β-actin, and presented relative to control (fold induction). Co-IP assays were carried out using the Pierce™ Classic Magnetic IP Kit (Thermo Fisher Scientific Inc., Waltham, MA) as described [49 (link)].

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Zheng S., Leclerc G.M., Li B., Swords R.T, & Barredo J.C. (2017). Inhibition of the NEDD8 conjugation pathway induces calcium-dependent compensatory activation of the pro-survival MEK/ERK pathway in acute lymphoblastic leukemia. Oncotarget, 9(5), 5529-5544.

Publication 2017

protease/phosphatase inhibitor cocktail BCA protein assay kit Bcl-2 Bcl-xL p-eIF2α STIM1 NEDD8 Pierce™ Classic Magnetic IP Kit

Actin Antibodies Assays Bcl 2 Buffer Camkii Chop Densitometry Erk1 Immunoblots Luciferase Mek1 Nedd8 Phosphatase Pkcα Pkcβ Protease inhibitor Proteins Ripa Stim1 Western blot

Corresponding Organization : Sylvester Comprehensive Cancer Center

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Variable analysis

independent variables

Treatment conditions (not explicitly specified)

dependent variables

Protein expression levels of ERK1/2, p-ERK1/2 (Thr202/Tyr204), MEK1/2, p-MEK1/2 (Ser217/221), p-PKCα/β II (Thr638/641), CAMKII, p-CAMKII (Thr286), BIM, p-BIM (Ser69), CHOP, Bcl-2, Bcl-xL, p-eIF2α (Ser51), STIM1, PARP, NEDD8
Protein-protein interactions (measured by co-IP assays)

control variables

Cell/tissue type (not explicitly specified)
Protein loading (normalized to β-actin)

positive controls

None specified

negative controls

None specified

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