20–30 μg of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. To minimize potential variability between analyses, all western blots were performed using aliquots of the same master preparation for each sample. Furthermore, to help ensure equal loading and proper transfer of proteins, all membranes were visually inspected using Ponceau-S staining immediately after transfer. Various proteins were targeted within whole cell lysates with primary antibodies against: Akt (9272; Cell Signaling Technology, Danvers, MA), pAktThr308 (9275; Cell Signaling Technology), GSK3β (9315; Cell Signaling Technology), pGSK3α/βSer21/9 (9331; Cell Signaling Technology), AS160 (ABS54; EMD Millipore, Billerica, MA), pAS160Thr642 (3028 P1; Symansis, Auckland, New Zealand), GPAT1 (4613; ProSci Incorporated, Poway, CA), DGAT1 (NB110–41487; Novus Biologicals, Littleton, CO), ATGL (2138; Cell Signaling Technology), CGI-58 (NB110–41576; Novus Biologicals), and HSL (4107; Cell Signaling Technology). Membranes were incubated with appropriate secondary antibodies and developed using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). Bands were imaged and then quantified via densitometry (AlphaEaseFC, Alpha Innotech Corp., Santa Clara, CA). Data are presented in arbitrary units relative to values obtained for 0mM (control) treated myotubes.
Pgsk3α βser21 9
Manufactured by Cell Signaling Technology
Sourced in United States
PGSK3α/βSer21/9 is an antibody that detects the phosphorylation of GSK3α at Ser21 and GSK3β at Ser9. GSK3 is a serine/threonine protein kinase that plays a key role in several cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Gsk3α β, by Cell Signaling Technology (3 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
Gsk3α β, by Santa Cruz Biotechnology (2 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
Cgi 58, by Novus Biologicals (1 mentions)
Leptin, by Thermo Fisher Scientific (1 mentions)
Pf573228, by Merck Group (1 mentions)
Anti icam 1, by Santa Cruz Biotechnology (1 mentions)
Sb216763, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Stattic, by Selleck Chemicals (1 mentions)
Ag490, by Merck Group (1 mentions)
Anti icam 1, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
P ampkα thr172, by Cell Signaling Technology (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
9 protocols using pgsk3α βser21 9
1
Western Blot Analysis of Insulin Signaling Proteins
2
Antibodies and Inhibitors for JAK-STAT Signaling
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Anti-p-JAK1 (Tyr1022/1023), p-JAK2 (Tyr1007/1008), p-FAK (Tyr397), and p-GSK3α/β (ser21/9) antibodies were purchased from Cell Signaling Technology (Danvers, MA, US). JAK1 and JAK2 were obtained from Elabscience (Houston, TX, US). U0126 was purchased from MCE (Monmouth Junction, NJ, US). JAK1 was purchased from Merck Millipore (Temecula, CA, US). Leptin was obtained from PeproTech (Rocky Hill, NJ, US). Anti-ICAM-1, ObR, FAK, p-ERK1/2, ERK, GSK3α/β antibodies, and ObR and control small interfering (si)RNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Stattic was purchased from Selleckchem (Houston, TX, US). AG490, PF573228, SB216763, anti-GAPDH, α-tubulin, and β-actin antibodies were obtained from Sigma-Aldrich (St. Louise, MO, US). Anti-p-GSK3β (Tyr216/279) and Anti-ICAM-1 antibodies were obtained from Thermo Scientific (Waltham, MA, US). Recombinant soluble ICAM-1 was purchased from KingFisher Biotech (Saint Paul, MN, US).
3
Evaluating Metabolic Pathway Regulators
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Primary antibodies were N‐cadherin (333900, Cell Signaling Technology Europe Invitrogen, Leiden, the Netherlands); AMPK‐α (Cell Signaling #2532); P‐AMPK‐α (Thr172) (40H9, Cell Signaling #2535); GSK‐3β (27C19, Cell Signaling #9315); P‐GSK‐3 α/β(Ser21/9) (Cell Signaling #9331); PKM2 (Cell Signaling #3198); H6PD (C‐10: sc‐377180). β‐actin or vinculin were used as a loading controls as indicated.
4
Western Blotting of Cell Fractions
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Western blotting was performed as per a previously described method.17 Primary antibodies against WDR41, AKT and β‐catenin were obtained from Santa Cruz Biotechnology (USA). p‐AKTser473, GSK‐3α/β and p‐GSK‐3α/βser21/9 antibodies were purchased from Cell Signalling Technology (Massachusetts, USA). GAPDH (Sangon Biotech, Shanghai, China) served as the internal control. For nuclear and cytoplasmic protein preparation, proteins were extracted using the nuclear and cytoplasmic extraction kit (CW0199S; CWBiotech, Beijing, China) and subjected to Western blotting to evaluate the differentially expressed proteins in the nuclear and cytoplasmic fractions. Analysis of grey scale intensity of the immunoblots was performed using Quantity One analysis software (Bio‐Rad, California, US).
5
Comprehensive Western Blotting Procedure for Protein Analysis
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The following antibodies were used for western blotting: DHX9 (Abcam, ab183731), GSK3α/β (Cell Signaling, #5676), p-GSK3α/β-Ser21/9 (Cell Signaling , #8566), P70S6K (Cell Signaling, #2708), p-P70S6K-Thr421/Ser424 (Cell Signaling, #9204), γH2AX(Cell Signaling, #9718), H2AX(Cell Signaling, #7631), p-Chk1 (Cell Signaling, #2348), Chk1 (Cell Signaling, #2360), Cyclin D2 (Cell Signaling, #3741), Myc (Cell Signaling, #5605), β-actin (Share-bio, SB-AB2001), secondary anti-rabbit IgG antibody (Proteintech, SA00001-2), secondary anti-mouse IgG antibody (Proteintech, SA00001-1). The detailed protocol is described in a previous study.8 (link)
6
Western Blot Analysis of Cellular Signaling
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Twenty micrograms of whole cell lysates were subjected to SDS-PAGE using BioRad 4–15% precast gels and wet transfer. Polyvinylidene difluoride (PVDF) membranes were probed with primary antibodies raised against the protein of interest; p-ACC(Ser79) (#3661, Cell Signaling Technology, Danvers, MA, USA), p-JNK(Thr183/Tyr185) (#9251, Cell Signaling Technology, Danvers, MA, USA), Hsp70 (#4872, Cell Signaling Technology, Danvers, MA, USA), p-Akt(Ser473) (#9271, Cell Signaling Technology, Danvers, MA, USA), CD36 (FAB19551A, R&D systems, Minneapolis, MN, USA), MyHC (Iowa Hybridoma Bank, Iowa City, IA, USA), p-GSK3 α/β(Ser21/9) (#9331 Cell Signaling Technology, Danvers, MA, USA), p-AS160(Thr642) (#4288 Cell Signaling Technology, Danvers, MA, USA). Detection of primary antibodies was performed using appropriate peroxidase-conjugated IgG and protein signals visualized using FEMTO enhanced chemiluminescence and BioRad Chemidoc XRS imager. Total protein content was quantified using reactive brown 10 (Sigma-Aldrich, St-Louis, MO, USA). Quantification of immunoblots was performed using ImageJ (NIH, Bethesda, MD, USA http://rsb.info.nih.gov/ij ).
7
ALS and Selumetinib Therapeutic Evaluation
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ALS and Selumetinib (Sel) were bought from Selleckchem Inc. (Houston, TX, USA) and stored at 100 mM in Dimethyl sulfoxide (DMSO) at -20°C. DMSO, fetal bovine serum (FBS), ammonium persulfate, protease and phosphatase inhibitor cocktails, doxycycline (DOX), and Dulbecco's phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St Louis, MO, USA). All required medium including EMEM, McCoy's 5A, RPMI1640, and DMEM were obtained from Corning Cellgro Inc. (Herndon, VA, USA). Pierce™ bicinchoninic acid (BCA) protein assay kit and radioimmunoprecipitation assay (RIPA) buffer were sourced from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Western blotting substrate was bought from Cell Signaling Technology (Beverly, MA, USA). Skim milk and nitrocellulose membrane were bought from BioRad (Hercules, CA, USA). Cleaved PARP, p-Akt (Ser473), Akt, p-Erk1/2 (Thr202/Tyr204), Erk1/2, p-GSK3α/β (Ser21/9), RAS, and LC3-I/II antibodies were all purchased from Cell Signaling Technology Inc. (Beverly, MA, USA), and β-actin was bought from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
8
Western Blot Analysis of AR-A014418 and Combination Treatments
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For Western blot experiments, cells were seeded into 10-cm plates and grown for 24 h in complete medium. Then, the medium was replaced by fresh medium, and cells were incubated with different concentrations of AR-A014418 (5 and 10 µM), either alone or in combination with 5-FU (5 µM) or everolimus (10 nM) . The incubation times were up to 96 h. Western blotting was conducted as described previously [43] . The following primary antibodies were used: pAktT (Ser473) (#4060), Akt (#2920), pERK1/2 (Thr202/Tyr204) (#4370), p4EBP1 (Ser65) (#9451), 4EBP1 (#9644), cyclin D3 (#2936), Chk1 (#2360), pEGFR (Tyr1068) (#3777), EGFR (#4267), β-catenin (#8480), pp70S6K (Thr389) (#9205), p70S6K (#2708), pS6 (Ser204/4) (#5364), S6 (#2217), pGSK3α/β (Ser21/9) (#9331), GSK3α/β (#5676) (Cell Signaling, Danvers, MA, USA), CgA (ab68271) (Abcam, Cambridge, UK), Bcl2 (610539) (Transduction Laboratories, USA), actin (A5441) (Sigma-Aldrich), ERK1/2 (06-182) (Merck-Millipore, Darmstadt, Germany).
9
Glycoside-induced Apoptosis Signaling
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Mahlavu cells were treated with either glycosides (2 μM) or DMSO control. 20-50 ng of protein was used per well (NuPAGE) for electrophoresis, which were then transferred to nitrocellulose membranes and blotted with specific antibodies (Cleaved caspase- 8 (9746S Cell Signaling),Akt (9272 Cell Signaling),Cruz), ERK1/2 (sc135900 Santa Cruz), SAPK/JNK (9252 Cell Signaling), pSAPK/JNK (Thr183/Tyr185) (4671 Cell Signaling), p-GSK3α/β (Ser21/9) (9331 Cell Signaling), GSK3-α/β (sc7291 Santa Cruz), Na + /K + -ATPase α1 subunit (3010 Cell Signaling), PARP (9532 Cell Signaling) and cleaved caspase-3 (9662S Cell Signaling). Antibodies were diluted in 1:100-500 5% BSA-TBS-T. Actin (Sigma, A5441), Calnexin (C4731 Sigma Aldrich) antibodies used for equal loading.
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