Cells were lysed in RIPA buffer complemented with Set I and Set II phosphatase inhibitors at 1× (Calbiochem), and protease inhibitors at 1× (Roche). Whole cell lysate concentration was determined with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Proteins were resolved on SDS-PAGE gels and electrotransferred to nitrocellulose membranes, 0.2 µm (Bio-Rad). Primary antibodies pS6S235/236, S6, pAKTS473, AKT, p4E-BP1T37/46, 4E-BP1, Cleaved PARP, pAktT308, p62, Rictor, Raptor, HIF-1α, HIF-2α, pERK1/2T202/Y204 (mouse), ERK, p-p90RSKS380, RSK1/2/3, pBADS112, pBADS136, pEGFRY1068, cleaved-caspase3 were from Cell Signaling Technologies. VHL (Santa Cruz #FL-181). mTOR primary antibody was from Millipore. Primary antibody dilutions were to manufactures' specifications (See Table S1 ). Tubulin (Sigma #T5168), KU-80 (GeneTex #GTX70485) and Actin-HRP (Santa Cruz #C-11) primary antibodies served as loading controls (LC) where noted. Secondary anti-Rabbit and ant-mouse antibodies were from (Fisher) and diluted in 5% milk, 1× TBS-T solution. ECL Western Blotting Detection reagents (GE Healthcare) were used for developing blots onto autoradiography film. For difficult to detect proteins SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used in combination with ECL.
Rsk1 2 3
Manufactured by Cell Signaling Technology
Sourced in United States
RSK1/2/3 is a family of serine/threonine protein kinases that are activated by the MAPK/ERK signaling pathway. These kinases play a role in the regulation of various cellular processes, including cell growth, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Phospho p90rsk, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Tubulin, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Pegfr y1068, by Cell Signaling Technology (1 mentions)
Hif 2α, by Cell Signaling Technology (1 mentions)
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
Ps6 s235 236, by Cell Signaling Technology (1 mentions)
P 4e bp1 t37 46, by Cell Signaling Technology (1 mentions)
P p90rsks380, by Cell Signaling Technology (1 mentions)
Pbads112, by Cell Signaling Technology (1 mentions)
Pbads136, by Cell Signaling Technology (1 mentions)
Actin hrp, by Santa Cruz Biotechnology (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
5 protocols using rsk1 2 3
1
Western Blot Analysis of Signaling Proteins
2
Comprehensive Cell Lysis and Immunoblotting
3
Western Blot Analysis of Signaling Pathways
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Cells were lysed using chilled RIPA buffer on ice. Protein concentration was determined using Bradford reagent and samples were run on a 4-12% Tris/Bis gel (Invitrogen). Protein was then transferred to an Immobilon-P PVDF membrane (Millipore), which was probed using antibodies against the following proteins: phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling 9101) ERK1/2 (Cell Signaling 9102), phospho-p90RSK (Ser380) (Cell Signaling 9341), RSK1/2/3 (Cell Signaling 9355), phospho-Akt (Ser473) (Cell Signaling 4058), Akt1/2/3 (Santa Cruz sc-81434), Ras (Cell Signaling 3965), E-cadherin (Invitrogen ECCD2) and GAPDH (Thermo Fisher MA5-15738) and then anti-mouse, anti-rabbit and anti-rat HRP-conjugated secondary antibodies (Dako). ECL detection was carried out using the Immobilon Crescendo HRP substrate (Millipore) in an ImageQuant LAS4000 (GE Healthcare).
4
Signaling Pathway Antibody Panel
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Phospho‐S6K1 (Thr389), phospho‐S6K1 (Thr421/Ser424), Akt, phospho‐Akt (Thr308), phospho‐Akt (Ser473), TSC1/Hamartin, phospho‐TSC2 (Ser939), phospho‐TSC2 (Ser1387), phospho‐TSC2 (Thr1462), phos‐ERK1/2 (Thr202/Tyr204), ERK1/2, phos‐ERK5 (Thr218/Tyr220), ERK5, phos‐rpS6 (Ser235/236), phos‐rpS6 (Ser240/244), rpS6, phos‐RSK (Thr359/Ser363), phos‐RSK (Ser380), and RSK1/2/3 were from Cell Signaling Technology (Danvers, MA, USA). TSC2/Tuberin (C‐20) and S6K1 (C‐18) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH, cadherin, myc, and phospho‐TSC2 (Ser664) were from Abcam (Cambridge, MA, USA). IRDye 800CW Goat anti‐(mouse IgG) and IRDye 680LT Goat anti‐(Rabbit IgG) secondary antibodies were from LI‐COR Biosciences.
5
Immunoblot Analysis of Protein Expression
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Vehicle or AP20187-treated cells were lysed using RIPA buffer containing 25 mM Tris HCl (pH 7.6), 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS and the protease inhibitor cocktail (Roche, Basel, Switzerland). Cell extracts with 5–20 μg of total protein were subjected to immunoblotting assays using primary antibodies against HA (3724, Cell Signaling Technology, Danvers, MA, USA), TNFAIP3 (sc-166,692, Santa Cruz Biotechnology, Dallas, TX, USA), E-cadherin (610,181, BD Biosciences, San Jose, CA, USA), N-cadherin (610,920, BD Biosciences), β-catenin (sc-7963, Santa Cruz Biotchnology), fibronectin (610,077, BD Biosciences), ERK1/2 (9102, Cell Signaling Technology), p-ERK1/2 (9101, Cell Signaling Technology), RSK1/2/3 (9355 s, Cell Signaling Technology), Phospho-p90 RSK (11,989 s, Cell Signaling Technology), GAPDH (2118 s, Cell Signaling Technology) and β-actin (A5441, Sigma-Aldrich, St., Louis, MO, USA). Appropriate horseradish peroxidase (HRP)-conjugated or fluorescence-labeled secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used to detect the primary antibodies bound to their antigens on the nitrocellulose membranes. The HRP activity was detected by using the ECL substrate solution (32,106, Thermo Fisher Scientific, Waltham, MA, USA), followed by exposure to X-ray film and quantified by the Odyssey Imaging System (LI-COR).
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