Proteomic Analysis of Cell Signaling

Cells were processed according to the previously described protocol [52 (link)] to obtain protein lysate that was electrophoresed through a reducing SDS 7.5, 10 or 15% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against S100A7 (36006), p-STAT3 (Y705), STAT3 (124H6), IGF-1R (3027S), pIGF-1R (Y1135/Y1136), ERK1/2 (9102S), pAKT (S473) D9E, AKT (C67E7), RAGE (D1A12), (all purchased from Cell Signaling Technology), pERK1/2 (E4), ERα (D-12) (Santa Cruz Biotechnology, DBA), and HA MMS-101R (Covance). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Cell Signaling, distributed by Euroclone, Milan, Italy) and revealed using the West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Life Technology, Milan, Italy). Chemiluminescent signal was revealed on Amersham high performance chemiluminescence films (Hyperfilms Amersham, distributed by Biogenerica, Catania, Italy), or using the LI-COR Odyssey 2800 (Li-COR Inc., Lincoln, NE, USA) and the software ImageStudioLite (version 5.2). β-actin (Sigma Aldrich, Merck, Milan, Italy) served as loading control. Original western blots and densitometric analyses of all blots shown are reported in Figures S3–S8.

Free full text: Click here

Muoio M.G., Talia M., Lappano R., Sims A.H., Vella V., Cirillo F., Manzella L., Giuliano M., Maggiolini M., Belfiore A, & De Francesco E.M. (2021). Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer. Cancers, 13(4), 621.

Publication 2021

ERK1/2 (9102S), pAKT (S473) D9E HA MMS-101R LI-COR Odyssey 2800 β-actin

Actin Antibodies Cells Chemiluminescence Densitometric Erk1 Horseradish peroxidase Igf 1r Membrane Nitrocellulose Polyacrylamide gel Proteins Rage S100a7 Stat3 Western blots

Corresponding Organization : University of Catania

Other organizations : University of Calabria, Edinburgh Cancer Research, Azienda Ospedaliero-Universitaria Policlinico - Vittorio Emanuele

Top 5 similar protocols

Variable analysis

independent variables

Protein lysate electrophoresed through a reducing SDS 7.5, 10 or 15% (w/v) polyacrylamide gel

dependent variables

Protein expression and phosphorylation levels of S100A7, p-STAT3 (Y705), STAT3 (124H6), IGF-1R, pIGF-1R (Y1135/Y1136), ERK1/2, pAKT (S473), AKT, RAGE, pERK1/2, ERα, and HA

control variables

β-actin (Sigma Aldrich, Merck, Milan, Italy) served as loading control

controls

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

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!