Immunoblotting Analysis of Cellular Signaling

Whole cell lysates were prepared and the immunoblotting was done as previously described [3 (link)] in Triton X 100-containing lysis buffer (50 mM Tris–HCl, pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF). For Western blotting, the following antibodies were used: pAKT (S473, #6942), AKT (#9272), Bcl-xL (#2764), p ERK1/2 (#9101), MEK1/2 (#4694), pMEK1/2 (#9121), ERK1/2 (#4696), NF-κB p65 (#6956), pNF-κB p65 (#3033), PARP (#9532), PI3K p85α (#13,666), pPI3K p85/p55 (#4228), PTK7 (#25618), Rac-1 (#4651), RhoA (#2117), ROR1 (#16,540), ROR2 (#88,639), Src (#2109), STAT3 (#9139), pSTAT3 (#9145) from Cell Signaling Technology (CST, Danvers, MA, USA); anti-pTYR 4G10 (#05–321) from Merck Millipore (Burlington, MA, USA); β-tubulin (#sc-166729) from Santa Cruz Biotechnology (Dallas, TX, USA); HA (#901,513) from BioLegend (San Diego, CA, USA). As secondary antibodies, IRDye^® 800CW Donkey anti-Mouse IgG or IRDye^® 680RD Donkey anti-Rabbit IgG (LI-COR, Lincoln, NE, USA) were used at 1:10 000 dilution.

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Raivola J., Dini A., Salokas K., Karvonen H., Niininen W., Piki E., Varjosalo M, & Ungureanu D. (2022). New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome. Cellular and Molecular Life Sciences, 79(5), 276.

Publication 2022

pAKT Bcl-xL pERK1/2 MEK1/2 pMEK1/2 ERK1/2 NF-κB p65 pNF-κB p65 PI3K p85α pPI3K p85/p55 STAT3 pSTAT3 anti-pTYR 4G10 β-tubulin IRDye® 800CW Donkey anti-Mouse IgG IRDye® 680RD Donkey anti-Rabbit IgG

Anti igg Antibodies Buffer Cell Dilution Donkey Edta Erk1 Glycerol Irdye 800cw Mek1 Mouse Nacl Nf κb p65 Pi3k Rabbit Rhoa Ror2 Stat3 Tris Triton x 100 Tubulin

Corresponding Organization :

Other organizations : University of Helsinki, Tampere University

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Variable analysis

independent variables

Triton X 100-containing lysis buffer (50 mM Tris–HCl, pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF)

dependent variables

Protein expression and phosphorylation levels of the following signaling proteins: AKT, Bcl-xL, ERK1/2, MEK1/2, NF-κB p65, PARP, PI3K p85α, PTK7, Rac-1, RhoA, ROR1, ROR2, Src, STAT3

control variables

β-tubulin (used as a loading control)

controls

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

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