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Phospho p90rsk

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Phospho-p90RSK is a laboratory reagent used to detect and quantify the phosphorylation of the ribosomal S6 kinase (RSK) protein. RSK is a serine/threonine protein kinase that plays a role in various cellular processes, including cell growth, proliferation, and survival. The Phospho-p90RSK product is used to monitor the activation state of RSK by measuring its phosphorylation levels.

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Lab products found in correlation

Phospho erk1 2, by Cell Signaling Technology (6 mentions) Erk1 2, by Cell Signaling Technology (4 mentions) Phospho akt, by Cell Signaling Technology (4 mentions) Phospho mek, by Cell Signaling Technology (3 mentions) P90rsk, by Cell Signaling Technology (2 mentions) Rsk1 2 3, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Phospho erk, by Cell Signaling Technology (2 mentions) β actin, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Complete mini, by Roche (1 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions) Phospho mek1 2, by Cell Signaling Technology (1 mentions) Mini trans blot cell and systems, by Bio-Rad (1 mentions) Mek1 2, by Cell Signaling Technology (1 mentions) West zol plus, by iNtRON Biotechnology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions) Erk1 2, by Merck Group (1 mentions)

Spelling variants of this product

P90RSK (18 citations) Polyclonal rabbit phospho-p90RSK (T359/S363) antibody (2 citations) Phospho-p90RSK (Ser380) (2 citations) Phospho-p90RSK (Thr359/Ser363) antibody (2 citations) Anti Phospho-p90RSK (S380) (2 citations)

17 protocols using phospho p90rsk

1

Hypoxic Stress Signaling Pathway

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The cells were seeded at a density of 5×105 cells per well and cultured in hypoxic conditions for 1 and 7 days. Then, they were lysed in a buffer containing 0.1% sodium dodecyl sulfate (SDS), 0.5 mM EDTA (pH 7.4), 1 mM Tri-HCl (pH 7.4), and protease inhibitor cocktail (Complete Mini, Roche Diagnostics, Mannheim, Germany). After treatment in two hypoxia-inducing systems for 3 days, nuclear and cytoplasmic proteins from cells were isolated using the NE-PER® Nuclear and cytoplasmic Extraction reagents (Thermo scientific, Rockford, IL, USA). The lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred onto 0.45-µM pore size polyvinylidene difluoride (PVDF, Pierce, Rockford, IL, USA) membranes using an electrophoretic transfer system (Mini Trans-Blot® Cell and systems, Bio-Rad, Hercules, CA, USA). The blots were incubated with antibodies against proteoglycan (Abcam®, Milton, UK), phospho-MEK1/2, MEK1/2, phospho-ERK1/2, ERK1/2, phospho-P90RSK, and P90RSK (Cell Signaling Technology® Inc, Danvers, MA, USA). After incubation with the secondary antibodies, immunoreactive bands were visualized using a Western blot detection system (West-Zol® plus, iNtRON Biotechnology, Seongnam, Korea). The blots were stripped of bound antibodies and reprobed using antibodies against actin (Abcam®) to verify the amounts of loaded protein.
Kang Y.M., Shin E.J., Lee B.H., Yang J.H., Lee H.M, & Moon S.H. (2021). Hypoxia Regulates the Extracellular Matrix via Mitogen-Activated Protein Kinases Pathway in Cells Retrieved from the Human Intervertebral Disc. Yonsei Medical Journal, 62(8), 734-742.
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2

Comprehensive Cell Lysis and Immunoblotting

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Cells were lyzed using 0.20% NP40, 50 mM Tris pH 7.5, 5% Glycerol, 1.5 mM MgCl2, 100 mM NaCl lysis buffer containing Phosphatase Inhibitor Cocktail 2 (Sigma, P5726) and cOmplete Protease Inhibitor Cocktail (Roche, 11873580001). Lysates were resolved by SDS-PAGE and incubated with primary antibodies. Antibodies used were against actin (A5441), CAMKK2 (HPA017389) (both Sigma), ERK1/2 (Sigma, M5670) and phospho-AKT (Ser473)(#9271), AKT (#9272), ALK(#3633), phospho-p90RSK (Ser380)(#12032), RSK1/2/3 (#9355), RSK1(#9333), RSK2 (#5528), phospho-RPS6 (Ser235/236)(#4858), RPS6 (#2217), phospho-ERK1/2 (Thr202/Tyr204)(#4267), phospho-FAK1 (Tyr397)(#8556), FAK1 (#13009), phospho-IGF1R (Tyr1131)(#3021), IGF1R (#9750), cleaved Caspase 3 (#9661), PARP-1 (#9542), AMPK1 (#2795), pAMPKα (Thr172)(#2535), FER (#4268), phospho-YB1 (Ser102)(#2900), and YB1 (#4202) were from Cell Signaling. Secondary antibodies were HRP-conjugated α-rabbit or α-mouse (GE Healthcare).
Kuenzi B.M., Remsing Rix L.L., Stewart P.A., Fang B., Kinose F., Bryant A.T., Boyle T.A., Koomen J.M., Haura E.B, & Rix U. (2017). Polypharmacology-based ceritinib repurposing using integrated functional proteomics. Nature chemical biology, 13(12), 1222-1231.
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3

Immunohistochemistry and Immunofluorescence Antibodies

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Antibodies utilized for immunohistochemistry and immunofluorescence were against smooth muscle actin (SMA;#M0851, DAKO, CA), MAC3 (#550292, BD, NJ), pan p90RSK (cat #MAB2056, Novus Biologicals, LLC, CO), and phospho-p90RSK (Ser380, # 9341) from Cell Signaling Technology (Danvers, MA).
Ko K.A., Wang Y., Kotla S., Fujii Y., Vu H.T., Venkatesulu B.P., Thomas T.N., Medina J.L., Gi Y.J., Hada M., Grande-Allen J., Patel Z.S., Milgrom S.A., Krishnan S., Fujiwara K, & Abe J.I. (2018). Developing a Reliable Mouse Model for Cancer Therapy-Induced Cardiovascular Toxicity in Cancer Patients and Survivors. Frontiers in Cardiovascular Medicine, 5, 26.
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4

Quantitative Protein Analysis in Cells

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Cells harvested at various times were lysed and analyzed using the BCA reagent to determine the protein concentration (Pierce, 23225), as we previously described [18 (link)]. 50–100 µg of protein was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane filter (Bio-Rad, 162-0177), and stained with Fast Green reagent (Thermo Fisher Scientific, BP123-10). Membrane filters were then blocked in 0.1 M Tris (pH 7.5)–0.9% NaCl–0.05% Tween 20 with 5% nonfat dry milk, and incubated with appropriate antibodies. Antibodies were diluted as follows: ATG7 (Cell Signaling, 8558), 1:2000; ERK1/2 (Cell Signaling, 4695), 1:2500; phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling, 9102), 1:2500; phospho-p90RSK (Thr359/Ser363, Cell Signaling, 9344), 1:2500; GAPDH (Cell Signaling, 2118), 1:5000; BiP (Cell Signaling, 3183), 1:2000; phospho-eIF2α (Ser51, Cell Signaling, 3398), 1:2000; SQSTM1 (Santa Cruz, sc-25575), 1:2000; CHOP/GADD153 (Santa Cruz, sc-575), 1:1000; anti-HA (Santa Cruz, sc-7392), 1:1000; LC3B (MBL International, PM036), 1:2000. The Supersignal West Pico (Pierce, 34077) and Femto chemiluminescence kits (Pierce, 34095) were used for visualization of the signal. Immunoblots were scanned and analyzed using Image Lab (BioRad, 170-9690).
Kim J.H., Hong S.K., Wu P.K., Richards A.L., Jackson W.T, & Park J.I. (2014). Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels. Experimental cell research, 327(2), 340-352.
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5

Co-localization of Protein Markers

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Paraffin-embedded slides were deparaffinized using xylene and ethanols. Antigen retrieval was performed using sodium citrate buffer, pH 6.0 containing 0.05% Tween-20. Blocking solution containing 10% goat serum diluted in Dako antibody diluent (Dako, Code S0809) were placed on slides for 1 h at room temperature. Primary antibody including phospho-p90RSK (Cell Signaling, 9341S), or CD34 (Abcam, ab8158) were used at 1:100 dilution and total p90RSK1 (Abclonal, A157118) at 1:50 dilution. 1:1000 goat anti-rabbit Alexa Fluor 647 (ThermoFisher, A27040) and 1:1000 goat anti-rat Texas Red (ThermoFisher, T6392) were used as secondary antibodies.
Images were captured with a Nikon Eclipse Ti de-convolution inverted fluorescent microscope.
Co-localization of fluorescence was quantified using Pearson's correlation coefficient in five random 20× magnification photographs per each slide and was averaged to obtain an average value for each tumor (Figures 8E,F).
Abe R.J., Savage H., Imanishi M., Banerjee P., Kotla S., Paez-Mayorga J., Taunton J., Fujiwara K., Won J.H., Yusuf S.W., Palaskas N.L., Banchs J., Lin S.H., Schadler K.L., Abe J.I, & Le N.T. (2020). p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway. Frontiers in Cardiovascular Medicine, 7, 542485.
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6

Activation of Cellular Signaling Pathways

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Dihydrocapsaicin was obtained from Cayman Chemical (Ann Arbor, MI, USA). Capsiate, capsanthin, capsaicin, 12-O-tetradecanoylphorbol 13-acetate (TPA), glutaraldehyde, crystal violet, glutamine, gentamicin, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Eagle’s MEM was purchased from Corning (New York, NY, USA). Antibodies for phospho-Akt, phospho-p38, phospho-JNK, phospho-p90RSK, phospho- phospho-p70S6K, phospho-S6, Akt, p70S6K, c-Fos, and p38 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies to detect phosphorylated ERK1/2, ERK1/2, and RSK2 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Lee J.S., Kim Y.A., Jang Y.J., Oh Y, & Byun S. (2019). Dihydrocapsaicin Inhibits Epithelial Cell Transformation through Targeting Amino Acid Signaling and c-Fos Expression. Nutrients, 11(6), 1269.
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7

Western Blot Protein Analysis

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For western blotting, cell lysates were first prepared using RIPA buffer (Sigma-Aldrich, St. Louis, MO) in accordance with the manufacturer’s instructions. Protein samples were then applied to the wells of NuPAGE 4%–20% Tris-Gly gel, electrophoresed in sodium dodecyl sulfate running buffer (Invitrogen), and transferred to nitrocellulose membranes using the iBlot transfer apparatus (Invitrogen). Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 (TBS-T) and 5% bovine serum albumin (BSA) for 1 hour at room temperature, followed by incubation with the primary antibody overnight at 4°C. On the following day, after the membranes were washed three times for 10 minutes each in TBS-T, horseradish peroxidase–conjugated secondary antibody (Bio-Rad, Hercules, CA) in TBS-T containing 2% BSA was applied for 1 hour at room temperature. Proteins were visualized with ECL Plus enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ) using G-box Chemi Systems (SynGene, Bengaluru, India). The commercial antibodies used in this study were ERK, phospho-ERK, MEK, phospho-MEK, AKT, phospho-AKT, c-Raf, tetraspanin 7 (TSPAN7), E-cadherin N-cadherin, cleaved poly(ADP-ribose) polymerase (PARP), PARP, ITGB3, ITGAv, HSP90, Phospho-p90RSK, RSK, phospho-FAK and β-actin (all purchased from Cell Signaling Technology).
Yoon S., Yang H., Ryu H.M., Lee E., Jo Y., Seo S., Kim D., Lee C.H., Kim W., Jung K.H., Park S.R., Choi E.K., Kim S.W., Park K.S, & Lee D.H. (2021). Integrin αvβ3 Induces HSP90 Inhibitor Resistance via FAK Activation in KRAS-Mutant Non–Small Cell Lung Cancer. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 54(3), 767-781.
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8

Protein Analysis of MAPK Pathway

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Cells harvested at various times were lysed in 62.5 mM Tris (pH 6.8)-2% SDS mixed with the protease inhibitor cocktail (Sigma-Aldrich) that contains 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin, and briefly sonicated before determining protein concentration using the BCA reagent (Pierce, Rockford, IL). 50 μg of protein was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane filter (Millipore, Billerica, MA), and stained with Fast Green reagent (Fisher Scientific, Pittsburgh, PA). Membrane filters were then blocked in 0.1 M Tris (pH 7.5)-0.9% NaCl-0.05% Tween 20 with 5% nonfat dry milk, and incubated with appropriate antibodies. Antibodies were diluted as follows: phospho-MEK1/2 (Ser217/221 for MEK1 and Ser222/226 for MEK2), 1:2,500; MEK1, 1:1,000; MEK2, 1:1,000; p21CIP1, 1:1,000 (Santa Cruz Biotech, Santa Cruz, CA); phospho-ERK1/2 (Thr202/Tyr204 for ERK1 and Thr183/Tyr185 for ERK2), 1:2,500; ERK1/2, 1:2,500; phospho-p90RSK (Thr359/Ser363), 1:2,500; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1:5,000 (Cell Signaling, Boston, MA); cyclin D1, 1:1,000 (Sigma-Aldrich). The Supersignal West Pico and Femto chemiluminescence kits (Pierce) were used for visualization of the signal. Images of immunoblots were taken and processed using ChemiDoc XRS+ and Image Lab 3.0 (BioRad, Hercules, CA).
Hong S.K., Wu P.K., Karkhanis M, & Park J.I. (2015). ERK1/2 can feedback-regulate cellular MEK1/2 levels. Cellular signalling, 27(10), 1939-1948.
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9

Ras Pathway Protein Detection Protocol

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Antibodies were obtained from the following sources: SOS1 (clone 25/SOS1), p120RasGAP (clone 13/RAS-GAP), MEK1 (no. 610121), MEK2 (no. 610235) were from BD Transduction Laboratories; K-Ras F234 (sc-30), N-Ras F155 (sc-31), pan-Ras C-4 (sc-166691), p-ERK1/2(Y204) (sc-101761), and Neurofibromin (sc-67) from Santa Cruz Biotechnology (Heidelberg, Germany); Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) (no. 2325), p44/42 MAPK (ERK1/2) (no. 4695), Akt (no. 9272), p-Akt(S473) (no. 4060), EGFR (no. 4267), p-EGFR (Y1068) (no. 2236), Phospho-p90RSK (Ser380) (no. 9341), RSK1/RSK2/RSK3 (32D7) (no. 9355), p-GSK-3β (Ser9) (D85E12) (no. 5558); GSK-3β (27C10) (no. 9315), Phospho-S6 Ribosomal Protein (Ser235/236) (no. 2211), S6 Ribosomal Protein (5G10) (no .2217), p70 S6 Kinase (49D7) (no. 2708) were from Cell Signaling Technology (Danvers, USA). Anti-DAB2IP (ab87811) was from Abcam (Cambridge, UK). Y13-259 rat monoclonal anti-Ras IP-antibody was purified from hybridoma supernatant (A.T.C.C., Manassas, U.S.A.).
Hennig A., Markwart R., Wolff K., Schubert K., Cui Y., Prior I.A., Esparza-Franco M.A., Ladds G, & Rubio I. (2016). Feedback activation of neurofibromin terminates growth factor-induced Ras activation. Cell Communication and Signaling : CCS, 14, 5.
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10

Western Blot Antibody Panel

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The Western blot method is described in detail in the Supplemental Material. The following antibodies were from Cell Signaling: GAPDH (8884), phospho-ERK1/2 (T202/Y204; 4370P), total ERK1/2 (4695P), phospho-AKT (S473; 4060P), total AKT (2920S), phospho-p90RSK (Ser380; 9341S), c-Myc (5605S), cyclin D1 (2978S), phospho-PRAS40 (Thr246; 2997S), KLF4 (4038S), phospho-Rb (Ser780; 3590S), total Rb (9309S), E2F1 (3742S), c-PARP (9541S), cleaved caspase substrate motif (8698S), phospho-RIP1 (Ser166; 65746S), total RIP1 (3493T), and LC3B (3868T). Other antibodies used were cyclin A (Santa Cruz Biotechonology, sc-751), vinculin (Sigma, V9131), and RRAS2 (Abcam, ab182264).
Liao S., Maertens O., Cichowski K, & Elledge S.J. (2018). Genetic modifiers of the BRD4-NUT dependency of NUT midline carcinoma uncovers a synergism between BETis and CDK4/6is. Genes & Development, 32(17-18), 1188-1200.
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