NHS-Rhodamine, Opti-MEM, anti-uPA antibody, and TRIzol reagent were purchased from Thermo Fisher Scientific. DAPI was obtained from Dojindo. Fibrinogen was purchased from Sigma-Aldrich. RSK inhibitor SL0101 and MEK inhibitor PD98059 were obtained from Merck Millipore. Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology. Anti-ERK1/2, and anti-MEK1/2 antibodies were purchased from Santa Cruz Biotechnology. LentiCRISPRv2, pMD2.G, and psPAX2 were obtained from Addgene. FuGENE HD was purchased from Promega. PrimeScript RT reagent Kit and SYBR Premix Ex Taq II were obtained from TaKaRa.
Anti rsk2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-RSK2 is a primary antibody that specifically recognizes the RSK2 protein. RSK2 is a serine/threonine-protein kinase that plays a role in the activation of the MAPK signaling pathway. This antibody can be used to detect and study the expression and localization of RSK2 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk, by Cell Signaling Technology (2 mentions)
Anti p rsk2, by Cell Signaling Technology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Fibrinogen, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Rsk inhibitor sl0101, by Merck Group (1 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti mek1 2, by Santa Cruz Biotechnology (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Nhs rhodamine, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Fugene hd, by Promega (1 mentions)
6 protocols using anti rsk2
1
Quantitative Analysis of Signaling Pathways
2
Protein Extraction and Western Blot Analysis
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As previously described [22 (link)], protein extraction was conducted using RIPA buffer (Sigma‐Aldrich, USA). Equal amounts of protein samples were separated using 12% SDS‐polyacrylamide gel electrophoresis. When the protein separation was complete, polyvinylidene difluoride membranes (Invitrogen, USA) were used for the protein transfer. Next, the samples were blocked in 5% skim milk (2 h). When the block was completed, anti-UBE2T (1;1000, Cat#: ab154022, Abcam, UK), anti-ERK, anti-p-ERK, anti-RSK2, anti-p-RSK2 (1:800; Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (1:1000; Abcam, Cambridge, MA), and anti‐GAPDH (1;1000, Cat#: ab8245, Abcam, UK) were used to treat the samples for overnight at 4°C. The next morning, the samples were treated with a secondary antibody (1 h). At the end, protein bands were observed with an Odyssey instrument (Li-cor, USA).
3
Immunofluorescent Staining of RSK2
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Cells were washed 3 times in pre-cooled phosphate buffered solution (PBS), fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, incubated with 0.2% Triton X-100 for 15 min, washed three times with PBS, and blocked for 30 min. Anti-RSK2 (Cell Signaling Technology, Danvers, MA, USA, 5528) diluted 1:500 was incubated at room temperature for 1 h, washed three times with PBS, and a specific secondary antibody was incubated at room temperature in the dark for 1 h and washed three times with PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (1 µg/mL; Roche, Switzerland) for 15 min at 37 °C and washed with PBS. Cells were mounted in a fluorescent mountant (Agilent, Santa Clara, CA, USA), and images were collected using a fluorescent microscope, counted with Image J software, and the number of RSK2-positive cells was calculated.
4
Protein Expression Profiling by Western Blot
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The extracted protein from cells was separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes at 100 mA for 2 h. The membranes were blocked in skimmed milk for 1 h at room temperature and overnight at 4°C in anti-CyclinD1, anti-Bcl-2, anti-Bax (1:1,000) and anti-ERK2, anti-p-ERK1/2, anti-RSK2, anti-p-RSK2, anti-MSK1 and anti-p-MSK1 (1:800; Cell Signaling Technology, Danvers, MA, USA), respectively, before they were conjugated with the secondary antibody. Signals were observed using Cano Scan (LiDE110, Tokyo, Japan).
5
Protein Expression Analysis Protocol
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Cells were lysed in RIPA buffer (Millipore, Billerica, MA, USA) containing protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA) and the obtained proteins (20 μg) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The proteins were identified using the following antibodies: anti-VGLL1 [2000:1, 10124-2-AP; Proteintech (Rosemont, IL, USA)]; anti-TEAD4 [2000:1, ab58310; Abcam (Cambridge, MA, USA)]; anti-integrin αV (1000:1, 4711S), anti-β-tubulin (3000:1, 2128S), anti-p-ERK (3000:1, 4370S), anti-ERK (3000:1, 4695S), and anti-RSK2 (2000:1, 5528S) (Cell Signaling Technology, Danvers, MA, USA); anti-GAPDH [5000:1, LF-PA0212; AB FRONTIER (Seoul, Korea)]; and anti-β-actin [3000:1, SC-47778; Santa Cruz Biotechnology (Dallas, TX, USA)]; anti-GFP [2000:1, MA5-15256; Thermo Fisher (Waltham, MA, USA)].
6
Immunofluorescence Staining of Neuronal Markers
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Sections were incubated in blocking solution (5% BSA, 1% Donkey Serum, and 0.5% Triton in DPBS) for at least 1 h at room temperature. Sections were then incubated overnight at 4°C with primary antibodies diluted in blocking solution: anti-Tuj1 (1/500, Biolegend), anti-RSK2 (1/100, Cell Signaling Technology), anti-RPS6 (1/100, Cell Signaling Technology), anti-p-RPS6Ser235-236 (1/100, Cell Signaling Technology), anti-p-RPS6Ser240-244 (1/500, Cell Signaling Technology), anti-RFP (1/500, Abcam), anti-SCG10 (1/1,000, Novus), anti-CTB (1/500, Abcam), anti-vGlut1 (1/1,000, Synaptic System), anti-vGat (1/500, Synaptic System), anti-ChAT (1/100, Merck), anti-Islet1-2 (5 μg/ml, DSHB), anti-Advillin (1/500, Proteintech), anti-TrkA (1/500, Bio-techne), anti-Parvalbumine (1/200, Swant), anti-TrkB (1/500, Bio-techne), anti-Calbindin (1/100, Swant), anti-Somatostatin (1/500, Invitrogen), and anti-PGP 9.5 (1/500, Proteintech). Then, tissues were incubated with the appropriated secondary antibodies (Alexa-Fluor conjugated—Jackson laboratories) diluted in blocking solution at 1/500 for 2 h at room temperature. Slides were mounted with Fluoromount-G Mounting Medium, with DAPI Medium (Invitrogen).
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