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PY416 is an antibody that specifically recognizes phosphorylation of tyrosine 416 in the activation loop of Src family kinases. This modification is associated with activation of these enzymes.

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Lab products found in correlation

Erk1 2, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Mglur1a, by Merck Group (1 mentions) Ps tp, by Abcam (1 mentions) Pxpsp, by Cell Signaling Technology (1 mentions) Mglur1a, by BD (1 mentions) P erk1 2, by Cell Signaling Technology (1 mentions) Phosphoserine, by Thermo Fisher Scientific (1 mentions) Actin, by Merck Group (1 mentions) Rs 3 5 dihydroxyphenylglycine dhpg, by Bio-Techne (1 mentions) D amphetamine sulfate, by Merck Group (1 mentions) Dpcpx, by Bio-Techne (1 mentions)

5 protocols using py416

1

Antibody-Based Investigation of Signaling Pathways

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Antibodies used in this study include rabbit antibodies against mGluR1a (Millipore; 1:2000), ERK1/2 (Cell Signaling; 1:2000), pERK1/2 (phosphorylation at T185/Y187 for pERK2; Cell Signaling; 1:1000), GST (Sigma; 1:2000), a phospho-serine/ threonine-proline motif (pS/TP, Abcam, Cambridge, MA; 1:1000), a phospho-MAPK motif PXpSP or pSPXR/K (Cell Signaling; 1:1000), phosphoserine (Invitrogen; 1:1000), phosphothreonine (Invitrogen; 1:1000), Src with phosphorylated tyrosine 416 (pY416, Cell Signaling; 1:1000), Src (Cell Signaling; 1:2000), or actin (Sigma; 1:4000) or mouse antibodies against mGluR1a (BD, Franklin Lakes, NJ; 1:2000), ERK1/2 (Cell Signaling; 1:2000), ERK2 (Abcam; 1:2000), or a phosphothreonine-proline motif (pTP, Cell Signaling; 1:1000). The anti-pY416 antibody reacts with the Src family members when phosphorylated at the conserved activation residue Y416. Pharmacological agents, including (S)-3,5-dihydroxyphenylglycine (DHPG), 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA), and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), were purchased from Tocris Cookson Inc. (Ballwin, MO). All drugs were freshly prepared at the day of experiments.
Yang J.H., Mao L.M., Choe E.S, & Wang J.Q. (2016). Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons. Molecular neurobiology, 54(9), 7156-7170.
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2

Immunoblotting of Glutamate Receptors

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We used the following primary antibodies: a mouse antibody against mGlu1a (BD Biosciences, San Jose, CA) or transferrin receptors (TfR, ThermoFisher), or a rabbit antibody against mGlu5 (MilliporeSigma), pY416 (Cell Signaling, Danvers, MA), Src (Cell Signaling), or β-actin (MilliporeSigma). Pharmacological agents used in this study include D-amphetamine sulfate (MilliporeSigma) and (RS)-3,5-dihydroxyphenylglycine (DHPG) (Tocris, Minneapolis, MN). At the day of experiments, all drugs were freshly prepared.
Mao L.M, & Wang J.Q. (2018). Amphetamine-induced conditioned place preference and changes in mGlu1/5 receptor expression and signaling in the rat medial prefrontal cortex. Neuroscience, 400, 110-119.
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3

Src Family Kinase Activation Assay

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Rabbit polyclonal antibodies used in this study include antibodies
against Src (Cell Signaling), Fyn (Santa Cruz Biotechnology, Santa Cruz, CA), or
β-actin (Sigma-Aldrich, St. Louis, MO). Mouse antibodies include those
against Src (Cell Signaling) or Fyn (Santa Cruz). A rabbit polyclonal antibody
against pY416 (Cell Signaling) was used. This antibody reacts with the Src
family members when phosphorylated at the conserved activation residue: Y416
(chicken Src), Y419 (rat Src), and Y420 (rat Fyn). Pharmacological agents
include (−)-scopolamine hydrobromide, SKF81297
[(±)-6-chloro-PB hydrobromide], and atropine sulfate.
These agents were purchased from Sigma-Aldrich (St. Louis, MO). All agents were
freshly prepared at the day of experiments. They were dissolved in physiological
saline.
Mao L.M., Faris H.J, & Wang J.Q. (2018). Muscarinic acetylcholine receptors inhibit Fyn activity in the rat striatum in vivo. Journal of molecular neuroscience : MN, 64(4), 523-532.
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4

Antibody Validation for Src Family Kinase Study

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Several primary antibodies were used in this study (Table 1). These include rabbit antibodies against Src (RRID:AB_2106047; Cell Signaling), Fyn (RRID:AB_631528; Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated Y416 (RRID:AB_10013641; pY416, Cell Signaling), or β‐actin (RRID:AB_476693; MilliporeSigma), or mouse antibodies against Src (RRID:AB_2106058; Cell Signaling), or Fyn (RRID:AB_627642; Santa Cruz). The anti‐pY416 antibody reacts with the Src family members when they are phosphorylated at the conserved Y416 site. Information on validation of antibodies is available from the companies. DPCPX was purchased from Tocris (Minneapolis, MN) and was dissolved in dimethyl sulphoxide and 0.1 M sodium hydroxide. Stock solutions were diluted in saline. The final concentration was 15% (v/v) for dimethyl sulphoxide and 8% (v/v) for sodium hydroxide (Uzbay et al., 2007 (link)). DPCPX was freshly prepared at the day of experiments.
Mao L, & Wang J.Q. (2021). Roles of adenosine A1 receptors in the regulation of SFK activity in the rat forebrain. Brain and Behavior, 11(8), e2254.
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5

Signaling Pathway Analysis Using Antibodies

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Rabbit or mouse primary antibodies were used. These antibodies include rabbit antibodies against Src (Cell Signaling Technology, Danvers, MA), Fyn (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated ERK1/2 (pERK1/2) at Thr202/Tyr204 (Cell Signaling), ERK1/2 (Cell Signaling), pY416 (Cell Signaling), or β-actin (Sigma-Aldrich, St. Louis, MO), or mouse antibodies against Src (Cell Signaling) or Fyn (Santa Cruz). A rabbit antibody against pY416 reacts with the Src family members when phosphorylated at the conserved activation residue: Y416 (chicken Src), Y419 (rat Src), and Y420 (rat Fyn). Pharmacological agents were purchased from Sigma-Aldrich (St. Louis, MO). These agents include D-amphetamine sulfate, R(+)-SCH23390 hydrochloride, SKF81297, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). All agents were freshly prepared at the day of experiments.
Jin D.Z., Mao L.M, & Wang J.Q. (2018). Amphetamine activates non-receptor tyrosine kinase Fyn and stimulates ERK phosphorylation in the rat striatum in vivo. European journal of pharmacology, 843, 45-54.
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