Immunoblot analysis of phospho-proteins

Immunoblot was performed as described previously [14 (link)]. 10 μg of protein per condition were used for SDS-PAGE analysis. Membranes were incubated with antibodies against phospho-RPS6 (Ser 240/244) (D68F8; Cell Signaling), phospho-RPS6 (Ser 235/236) (D57.2.2.E; Cell Signaling), phospho-PRAS40 (Thr246) (C77D7, Cell signaling) or actin (# sc-1616 Santa Cruz Biotechnology, Dallas, Texas, USA). The secondary HRP-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). A chemiluminescence solution was used for detection [50 (link)].

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Ronellenfitsch M.W., Zeiner P.S., Mittelbronn M., Urban H., Pietsch T., Reuter D., Senft C., Steinbach J.P., Westphal M, & Harter P.N. (2018). Akt and mTORC1 signaling as predictive biomarkers for the EGFR antibody nimotuzumab in glioblastoma. Acta Neuropathologica Communications, 6, 81.

Publication 2018

phospho-RPS6 (Ser 240/244) (D68F8; phospho-RPS6 (Ser 235/236) (D57.2.2.E;

Actin Antibodies Chemiluminescence Immunoblot Membranes Protein Sds page

Corresponding Organization : Goethe University Frankfurt

Other organizations : University of Bonn, Oncoscience (Germany), Frankfurt Cancer Institute, Universität Hamburg

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Variable analysis

independent variables

None mentioned

dependent variables

Phosphorylation of RPS6 (Ser 240/244)
Phosphorylation of RPS6 (Ser 235/236)
Phosphorylation of PRAS40 (Thr246)

control variables

Protein amount (10 μg per condition)
Antibodies used (phospho-RPS6, phospho-PRAS40, actin)
Secondary HRP-conjugated antibodies
Chemiluminescence detection method

positive controls

None mentioned

negative controls

None mentioned

Annotations

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