Protein Extraction and Western Blot Analysis

Cells were homogenized in RIPA buffer as described^{17 (link)}. Nuclear and cytoplasmic extracts were isolated by centrifugation and hypotonic lysis as described^{18 (link)}. Antibodies for analyses included: RON (SC-322), Androgen Receptor, (SC-815), Axl (SC-1096), Gas6 (SC-1935), and Tubulin (SC-5286) from Santa Cruz Biotechnology; Src (2110S), phosphor-Src y416 (2101S), Akt (4691S), phosphor-Akt s473 (4060S), phospho-Axl y702 (5724S) and LAMIN A/C (4777S) from Cell Signaling Technologies; phospho-RON y1238/y1239 (AF1947, R&D); ACTIN (Cincinnati Children’s Hospital Medical Center, Clone C4). Peroxidase-conjugated secondary antibodies (Jackson Laboratories) were applied, and membranes were developed using Pierce ECL2 Western Blotting substrate (ThermoFisher Scientific). Membranes were stripped using Restore Western Blot Stripping Buffer (ThermoFisher Scientific) before re-probing. The Proteome Profiler Mouse Phospho-RTK Array Kit (ARY014, R&D) was used on whole tumor lysates and performed according to manufacturer’s instructions.

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Brown N.E., Jones A., Hunt B.G, & Waltz S.E. (2022). Prostate tumor RON receptor signaling mediates macrophage recruitment to drive androgen deprivation therapy resistance through Gas6‐mediated Axl and RON signaling. The Prostate, 82(15), 1422-1437.

Publication 2022

Tubulin (SC-5286) LAMIN A/C (4777S) phospho-RON y1238/y1239 (AF1947, Peroxidase-conjugated secondary antibodies Pierce ECL2 Western Blotting substrate Restore Western Blot Stripping Buffer Proteome Profiler Mouse Phospho-RTK Array Kit

Actin Androgen receptor Antibodies Buffer Cells Centrifugation Clone Cytoplasmic Lamin a c Membranes Mouse Peroxidase Phosphor Proteome Ripa Tubulin Tumor Western blot

Corresponding Organization : Veterans Health Administration

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Variable analysis

independent variables

Homogenization in RIPA buffer
Isolation of nuclear and cytoplasmic extracts by centrifugation and hypotonic lysis

dependent variables

Protein expression and phosphorylation levels of RON, Androgen Receptor, Axl, Gas6, Src, Akt, Axl, and LAMIN A/C
Phospho-RTK signaling profile using Proteome Profiler Mouse Phospho-RTK Array Kit

control variables

Tubulin
ACTIN

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

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