Preparation of whole-cell extracts and immunoblotting were done essentially as described [32 (link)]. Briefly, protein lysates were separated on NuPAGE 4–12% Bis-Tris gradient gels (Life Technologies) using NuPAGE MOPS SDS Running Buffer (Life Technologies) and transferred by semi-dry blotting onto polyvinylidene diflouride membrane (GE Healthcare). Equal loading and transfer were confirmed by Amido Black staining (Sigma Aldrich). All washing and incubation steps were carried out with Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat dry milk or BSA. Primary antibodies used were: AKT (#9272), phospho-CREB (Ser133) (#9198), phospho-GSK3α (Ser21) (#9316), phospho-GSK3β (Ser9) (#5558), phospho-p38 MAPK (Thr180/Tyr182) (#9211), phospho-MKK3/6 (Ser189/Ser207) (#12280), phospho-HSL (Ser660) (#4126), phospho-HSL (Ser563) (#4139), phospho-(Ser/Thr) PKA substrate (#9621) (all from Cell Signaling Technology), TFIIB (#sc-225) (Santa Cruz Biotechnology), CYC1 (#sc-7159) (Santa Cruz Biotechnology), FABP4 (#10004944) (Cayman Chemical) and UCP1 (#10983) (Abcam). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit or anti-mouse (DAKO). EZ-ECL Enhanced Chemiluminescence Detection Kit for HRP (Biological Industries) was used for detection.
Phospho ser thr pka substrate
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Czechia
Phospho-(Ser/Thr) PKA Substrate is a lab equipment product that detects and identifies proteins containing PKA (Protein Kinase A) phosphorylation sites. It is used to study the activity and regulation of PKA, a key enzyme involved in various cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (2 mentions)
Amido black staining, by Merck Group (2 mentions)
Ez ecl enhanced chemiluminescence detection kit for hrp, by Sartorius (2 mentions)
Tfiib, by Santa Cruz Biotechnology (2 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (2 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Phospho creb ser133, by Cell Signaling Technology (1 mentions)
Phospho hsl ser660, by Cell Signaling Technology (1 mentions)
Fabp4, by Cayman Chemical (1 mentions)
Phospho hsl ser563, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pgc 1a, by Merck Group (1 mentions)
P38 mapk d13e1 xp, by Cell Signaling Technology (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
11 protocols using phospho ser thr pka substrate
1
Immunoblotting Analysis of Cellular Signaling
2
Western Blot Analysis of Cellular Proteins
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Total protein from tissues or cells was prepared with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche). The protein samples were boiled for 10 min and then were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore). Next, the membranes were blocked with 5% milk for 1 h at room temperature and incubated with the corresponding antibodies against UCP1 (Abcam), PGC-1a (Millipore), β-Actin (Cell Signaling Technology), AMPK (Cell Signaling Technology),Phospho-AMPKα (Thr172) (Cell Signaling Technology), p38 MAPK (D13E1)XP® (Cell Signaling Technology),Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), Phospho- (Ser/Thr) PKA Substrate (Cell Signaling Technology), Tubulin (Sigma) and IRF4 (Santa Cruz) overnight at 4 °C. Subsequently, the membranes were incubated with the secondary antibody for 1 h at room temperature. An ECL detection system was used to detect the signals (GE Healthcare, USA).
3
Immunohistochemical Analysis of MAPK and PKA
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Adjacent slides from FFPE blocks were stained for H&E or incubated with primary antibodies against Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) or Phospho-(Ser/Thr) PKA Substrate (Cell Signaling Inc.). All staining was compared to normal appendix. The signal was scored by staining intensity (0 to 3+) and fraction of stained tumor cells (0% to 100%). The enzyme was considered active at a signal greater than 2 in more than 25% of tumor cells.
Complete material and methods are available as Supplemental Information (Additional file2 ).
Complete material and methods are available as Supplemental Information (Additional file
4
Immunoblotting of Protein Signaling Pathways
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Preparation of whole-cell extracts and immunoblotting were carried out essentially as described55 (link). Briefly, protein lysates were separated using NuPage 4–12% Bis-Tris gradient gels (Life Technologies) and transferred by semi-dry blotting onto polyvinylidene difluoride membrane (GE Healthcare). Equal loading was confirmed by Amido Black staining (Sigma-Aldrich). All washing and incubation steps were carried out with Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat dry milk or BSA. Primary antibodies used were: CREB (#9192), p-CREB (Ser133) (#9198), GSK3α (#4337), p-GSK3α (Ser21) (#9316), GSK3β (#12456), p-GSK3β (Ser9) (#5558), p38 MAPK (#9212), p-p38 MAPK (Thr180/Tyr182) (#9211), MKK3 (#8535), MKK6 (#8550), p-MKK3/6 (Ser189/Ser207) (#12280), ATF2 (#9226), p-ATF2 (Thr71) (#5112), HSL (#4107), p-HSL (Ser660) (#4126), Phospho-(Ser/Thr) PKA substrate (#9621) (all from Cell Signaling Technology), TFIIB (#sc-225) (Santa Cruz Biotechnology), Vinculin (#V9264) (Sigma-Aldrich), UCP1 (#10983) (Abcam) and HA (#11583816001) (Roche). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit or anti-mouse (DAKO). EZ-ECL Enhanced Chemiluminescence Detection Kit for HRP (Biological Industries) was used for detection.
5
Immunoblotting Analysis of Protein Phosphorylation
6
Liver Protein Expression Analysis
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Whole‐cell lysates were extracted from liver, and 20 µg of liver extracts were separated on polyacrylamide gel and transferred to a nitrocellulose membrane. Target proteins were detected by western blot and immunostaining with specific primary antibody, followed by horseradish peroxidase–labeled secondary antibody. The specific immunoreactive bands of interest were detected by chemiluminescence (Bio‐Rad). Digital system (ImageQuant LAS 4000; GE Healthcare) was used for image acquisition. Antibodies were specific for Fatty Acid Synthase (3180S, Cell Signaling Technology), Phospho‐(Ser/Thr) PKA Substrate (9621S, Cell Signaling Technology), CYP7A1 (MABD42, EMD Millipore), and Casp1p10 (sc‐514, Santa Cruz Technology).
7
Dopaminergic and Cholinergic Neurotransmission
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SKF 81297 and quinpirole were from Tocris (Minneapolis, MN, USA), dicyclomine, 6-OHDA-HCl, L-DOPA (3-(3,4-Dihydroxyphenyl-2,5,6-d3)-L-alanine), desipramine, pargyline and benserazide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-tyrosine hydroxylase (RRID: AB_390204) and anti-Choline Acetyltransferase (RRID:AB_2079751) and anti-pS10H3 (RRID:AB_1977177) were purchased from Millipore. Anti-phospho-Thr202/Tyr204–Erk1/2 (RRID:AB_331646), and phospho-(Ser/Thr) PKA substrate (RRID:AB_331817), a-DARPP-32 (RRID:AB_823479).
And anti pS6 ribosomal protein (RRID:AB_916156) were from Cell Signaling Technology. Various Alexa Fluor antibodies were used at a 1:500 dilution for immunohistochemistry (Invitrogen).
And anti pS6 ribosomal protein (RRID:AB_916156) were from Cell Signaling Technology. Various Alexa Fluor antibodies were used at a 1:500 dilution for immunohistochemistry (Invitrogen).
8
Protein Extraction and Western Blot
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Cells were harvested and disrupted in a buffer containing 50mM HEPES pH 7.5, 150mM NaCl, 1% (v/v) glycerol; 1% (w/v) Triton X-100, 1.5mM MgCl2, 5mM EGTA, protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (Sigma-Aldrich). 10 to 30μg of total protein were resolved by SDS-PAGE and transferred to the nitrocellulose membrane, which was incubated overnight with specific antibodies: vinculin, Grp78, CHOP (GADD153) IDH1 and GSS from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); phospho-(Ser/Thr) PKA Substrate, phospho-CREB S133, total CREB, Beclin-1, LC3, Eif2α, cleaved caspase 3, phospho-Src (Y416) and total Src (36D10) from Cell Signaling Technology Inc. (Danvers, MA, USA); O-Linked N-Acetylglucosamine (Clone RL2) from Thermo Scientific (Waltham, MA, USA).
9
Mitochondrial Function and Oxidative Stress Assays
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MitoTracker Green, MitoTracker DeepRed, MitoSox, and CellRox were purchased from Molecular Probes. Proteinase K was purchased from Roche. Recombinant human GM-CSF was purchased from BD. Recombinant human LL-37, R837, ODN-TTAGGG, and ODN-2216 were purchased from InvivoGen. FcR Blocking Reagent was purchased from Miltenyi Biotec. Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3-phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam. Antibodies against MnSOD were purchased from Millipore. Antibodies against HMGB1, PGC1α, and Phospho-(Ser/Thr) PKA Substrate were purchased from Cell Signaling Technologies. Genomic DNA was obtained from BioChain. IRS661 and DVX41 were a gift from Dynavax Technologies Corporation. Specified neutrophils were cultured in the presence of αRNP-IgG (50 µg/ml), Recombinant human GM-CSF (50 ng/ml), diphenylene iodonium (DPI; 10 µM), MT (10 µM), CCCP (25 µM), Rotenone (5 µM), Oligomycin (10 µM) plus Antimycin (1 µM; O+A), IRS661 (TLR7 antagonist; 1 µM), DVX42 (TLR8 antagonist; 1 µM), Bafilomycin A1 (BafA1; 1 µM), 8Br-cAMP (100 µM), IBMX (50 µM), or H89 (1 µM).
10
Western Blot Analysis of Cellular Proteins
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Cytosolic, total or immunoprecipitated proteins were separated by electrophoresis on SDS-polyacrylamide gels [44 (link)], and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Perkin Elmer Life Sciences, Boston, MA, USA). Membranes were blocked with 5% non-fat milk/0.3% Tween/PBS, washed and incubated overnight with specific primary antibody [cytochrome c (Santa Cruz Biotechnology Inc.); α tubulin (Sigma Chemical Co.); β actin (Sigma Chemical Co.); phospho-(Ser/Thr) PKA Substrate (Cell Signaling Technology); p21 (Santa Cruz Biotechnology Inc.); AMPKα (Cell Signaling Technology); Phospho-AMPKα (Thr172) (Cell Signaling Technology); Phospho-AMPKα1/2 (Ser173) (Abcam, Cambridge, UK); Puma (Santa Cruz Biotechnology Inc.)]. Membranes were washed and probed with the appropriate secondary antibody conjugated to horseradish peroxidase. Bands were detected by chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ), after exposition to Kodak XAR film. Bands were quantified using the Image J software. In preparing the figures, brightness and contrast were adjusted in order to improve visualization.
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