Kinase activity was measured at 37 °C for 30 minutes in 50 µl kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl2) supplemented with 50 µM ATP and human recombinant FOXO1 (1 µg; Origene #TP300477). Kinase reactions were run on 4–20% Tris-Glycine polyacrylamide gels (Thermofisher) and byproducts identified with anti phospho-Serine/Threonine antibody (Abcam ab17464).
Ab17464
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab17464 is a primary antibody that specifically recognizes and binds to the target antigen. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab2254, by Abcam (1 mentions)
Ab3446, by Abcam (1 mentions)
Ab2254, by Proteintech (1 mentions)
Magnetic bead, by Thermo Fisher Scientific (1 mentions)
Direct magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Transgene (1 mentions)
B900610, by Proteintech (1 mentions)
Ab4729, by Abcam (1 mentions)
H3k27ac, by Abcam (1 mentions)
Smad1 5 8, by Santa Cruz Biotechnology (1 mentions)
Ampkα1, by Cell Signaling Technology (1 mentions)
P smad1 5 8, by Cell Signaling Technology (1 mentions)
Ampkα2, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Ab51253, by Abcam (1 mentions)
Ab184674, by Abcam (1 mentions)
Ab80627, by Abcam (1 mentions)
Ab3416, by Abcam (1 mentions)
Mab5280, by Merck Group (1 mentions)
9 protocols using ab17464
1
FOXO1 Kinase Activity Assay
2
Protein Extraction and Co-Immunoprecipitation
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Total protein was extracted with RIPA lysate, and cytoplasmic/nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech). After determining the concentration by the BCA method, proteins were heated at 99°C for 10 min, subjected to SDS‐PAGE gel electrophoresis, and transferred to the PDVF membrane. Immunoblots were carried out with primary antibodies, including anti‐NFAT5 (Abcam, ab3446), anti‐AURKB (Abcam, ab2254), anti‐Phospho‐Ser/Thr (Abcam, ab17464), anti‐AQP4 (Abcam, ab2254), anti‐GAPDH (ZENBIO, 20030–67E4), anti‐Histone 3 (CST, 4499s).
Co‐IP assay was performed using a Direct Magnetic IP/CO‐IP Kit (Thermo Fisher Scientific, 88 824). Briefly, rat SDH tissue was lysed with IP lysis buffer supplied with protease and phosphatase inhibitors, and then incubated with the primary antibodies: anti‐NFAT5 (Novus Biologicals, NB1203446), or anti‐AURKB (Abcam, ab2254), or anti‐IgG (Proteintech, B900610). Magnetic beads (Thermo Fisher Scientific, 88 824) were used to pull down the corresponding protein complex. After eluted the proteins from the beads, and western blot was used to identify the specific protein.
Co‐IP assay was performed using a Direct Magnetic IP/CO‐IP Kit (Thermo Fisher Scientific, 88 824). Briefly, rat SDH tissue was lysed with IP lysis buffer supplied with protease and phosphatase inhibitors, and then incubated with the primary antibodies: anti‐NFAT5 (Novus Biologicals, NB1203446), or anti‐AURKB (Abcam, ab2254), or anti‐IgG (Proteintech, B900610). Magnetic beads (Thermo Fisher Scientific, 88 824) were used to pull down the corresponding protein complex. After eluted the proteins from the beads, and western blot was used to identify the specific protein.
3
Validation of Antibodies for Dyrk1a
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Antibodies against Actin, Tubulin and Flag were from Sigma, CBP from Diagenode (C15410224) for western blots, Novus (NB100-381) and ThermoFisher (PA5-27369) for ChIP, p300 from Santa Cruz (SC585) or from Invitrogen (PA1-848), H3K27ac was from Abcam (ab4729). Pan-phospho antibody was from Abcam (ab17464). Rabbit antisera were raised against two different Dyrk1a peptides—1598-HSHQYSDRRQPNISDQQVSALSYSD QIQQPLTNQ, 1602-TYQFSANTGPAHYMTEGHLTM RQGADREES. Antibodies were validated by shRNA mediated knockdown of DYRK1A (Supplemental Figure S1 ).
4
Antibody-based Protein Detection Assays
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Western blots and immunohistochemical staining were performed as described.28 , 29 , 30 Antibodies used were SLC40A1 (NBP1‐21502, Novus Biologicals, Centennial, CO, USA, 1:500), GAPDH (sc‐32233, Santa Cruz Biotechnology, Dallas, TX, USA, 1:2000), AMPKα1 (2795, Cell Signaling Technology, Danvers, MA, USA, 1:1000 and sc‐19128, Santa Cruz Biotechnology, 1:500), AMPK‐α2 (2757, Cell Signaling Technology, 1:1000 and sc‐19131, Santa Cruz Biotechnology, 1:1000), pAMPK (Thr172, 2535, Cell Signaling Technology, 1:1000), Smad1/5/8 (sc‐6031, Santa Cruz Biotechnology, 1:1000), pSmad1/5/8 (9511, Cell Signaling Technology, 1:1000), HIF1α (14179, Cell Signaling Technology, 1:1000), Hydroxy‐HIF1α (Pro564) (3434, Cell Signaling Technology, 1:1000), pSer/Thr (ab17464, Abcam, 1:1000 ), PHD2 (4835, Cell Signaling Technology, 1:1000), HA (3724, Cell Signaling Technology, 1:1000) and His (12698, Cell Signaling Technology, 1:1000).
5
Investigating α-Synuclein Phosphorylation Pathways
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The primary antibodies and chemicals used were as follows: rabbit anti-TH (Pel-Freez, Brown Deer), mouse anti-TH (MAB5280, Millipore), rabbit anti-phospho-PAK4 (S474) (#3241, Cell Signaling), rabbit anti-PAK4 (ab19007, Abcam), mouse anti-α-synuclein (SNCA) (ab80627, Abcam), mouse anti-α-synuclein (SNCA) (MAB5383, Abnova), rabbit anti-phospho-α-synuclein (S129) (SNCA) (ab51253, Abcam), mouse anti-phospho-α-synuclein (S129) (SNCA) (ab184674, Abcam), Anti-phospho-serine/threonine (ab17464, Abcam), anti-phospho-tyrosine (05-321, Upstate), rabbit anti-GAPDH (sc25778, Santa Cruz Biotechnology), mouse anti-Parkin (#4211, Cell Signaling), rabbit anti-Chip (#2080, Cell Signaling), goat anti-Siah-1 (ab2237, Abcam), rabbit anti-NEDD4-1 (#2740, Cell Signaling), rabbit anti-NEDD4-2 (ab46521), rabbit anti-GFP (sc8334, Santa Cruz Biotechnology), rabbit anti-Myc (sc789, Santa Cruz Biotechnology), rabbit anti-His (#2366S, Cell Signaling), and GST-conjugated horseradish peroxidase (HRP) (ab3416, Abcam), tamoxifen (T5648, Sigma-Aldrich), Heclin (SML1396, Sigma-Aldrich), indole-3-carbinol (I7256, Sigma-Aldrich), Nutlin3a (SML0580, Sigma-Aldrich), phosphatase inhibitor cocktails II (P5726, Sigma-Aldrich) and III (P0044, Sigma-Aldrich) and complete protease inhibitor mixture (#5871, Cell Signaling Technology, Danvers, MA, USA).
6
Kinase Phosphorylation Assay Protocol
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For in vitro assays of kinase phosphorylation, the dephosphorylated kinases were mixed and incubated in a 20-μL reaction buffer containing 20 mM HEPES (pH 7.3), 150 mM NaCl, 10 mM MgCl2, 0.2 mM EDTA, and 1 mM ATP. All incubation reactions were performed at 25°C. Samples were collected at specific time points, mixed with 2 μL of loading buffer, and heated at 100°C for 2 min. All protein samples were then separated on 10% SDS–PAGE gels and stained with Coomassie brilliant blue. For western blotting analysis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) and blocked using 5% BSA solution prepared in TBST buffer. The membranes were incubated with an anti-phospho-Ser/Thr antibody (1:3300; Abcam, ab17464, Cambridge, UK) for 12 h at 4°C, then incubated with peroxidase-coupled goat anti-rabbit antibody (1:5000; Abcam, ab6721, Cambridge, UK) for 4 h at 4°C. Finally, the membranes were subjected to enhanced chemiluminescence detection (Yamei, SQ201, Shanghai, China) to visualize and monitor the phosphorylation status of the proteins. The original blot and SDS–PAGE images can be found in Supplemental Figure 13 .
7
KRIT1 Phosphorylation in HeLa and HPAECs
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GFP–KRIT1-transfected HeLa cells treated with PMA or BIM+PMA were lysed in NP-40 buffer (Sigma) containing protease and phosphatase inhibitors (P8340 and P2850, respectively; Sigma). GFP–KRIT1 was immunoprecipitated from cell lysates using the rabbit polyclonal anti-GFP antibody (ab290, Abcam), and analyzed by western blotting with pan-phospho-Ser/Thr antibody ab17464 (1:1000). Western blotting analysis was performed as previously described (Balzac et al., 2005 (link)).
mCherry–KRIT1-expressing HPAECs treated with PMA, BIM and BIM+PMA were lysed in buffer containing 20 mM HEPES-KOH pH 7.5, 1.5 mM MgCl2, 5 mM KCl, and protease and phosphatase inhibitors, supplemented with 1% Triton X-100. KRIT1 was immunoprecipitated (Mab15.B2, Millipore, Burlington, MA) from total lysate and blotted with pan-phospho-Ser/Thr antibody 22A (1:1000).
mCherry–KRIT1-expressing HPAECs treated with PMA, BIM and BIM+PMA were lysed in buffer containing 20 mM HEPES-KOH pH 7.5, 1.5 mM MgCl2, 5 mM KCl, and protease and phosphatase inhibitors, supplemented with 1% Triton X-100. KRIT1 was immunoprecipitated (Mab15.B2, Millipore, Burlington, MA) from total lysate and blotted with pan-phospho-Ser/Thr antibody 22A (1:1000).
8
Antibody-based Protein Analysis Protocol
9
ZRANB1 Kinase Activity Assay
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Briefly, the assay was performed in a total volume of 50-μl reaction mixture. One μg recombinant ZRANB1 or ZRANB1 mutant protein or UVRAG or its mutant protein was incubated with 1 μg CSNK1A1. The kinase buffer contains 1 mM DTT, 10 μM cold ATP, 1 mM Na3VO4, 10 mM MgCl2, 50 mM HEPES, pH 7.4, and 5 μCi [γ-32P]-ATP for 1 h at 30°C. Then the reaction system was stopped by the addition of 10 μl 4× sample buffer and probed with phospho-specific antibodies (Abcam, ab17464).
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