- PA5-78123 - Invitrogen Antibodies PRKAR2A antibody

Submitted references Tau inhibits PKA by nuclear proteasome-dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation.

Ye J, Yin Y, Liu H, Fang L, Tao X, Wei L, Zuo Y, Yin Y, Ke D, Wang JZ
Aging cell 2020 Jan;19(1):e13055

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: PRKAR2A Polyclonal Antibody detects PKA R2 protein by Western blot analysis. A. 30 µg 293T whole cell lysate/extract. B. 30 µg A431 whole cell lysate/extract. C. 30 µg HeLa whole cell lysate/extract. D. 30 µg HepG2 whole cell lysate/extract.10 % SDS-PAGE. PRKAR2A Polyclonal Antibody (Product # PA5-78123) dilution: 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: PRKAR2A antibody detects PRKAR2A protein at cytoplasm by immunofluorescent analysis. Sample: HeLa cells were fixed in 100% MeOH for 5 min. Green: PRKAR2A protein stained by PRKAR2A antibody (Product # PA5-78123) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Overexpressing hTau inactivates PKA with proteasome-involved elevation of PKAR2alpha and inhibition of CREB/BDNF signaling. (a) Overexpressing hTau inhibits PKA without changing cAMP in cultured hippocampal neurons (5 + 7 div ) measured, respectively, by using PKA activity assay kit and the cAMP ELISA kit. (b, c) Overexpressing hTau inhibits PKA activity with an elevated PKAR2alpha in nuclear fraction of the cultured neurons. The hippocampal neurons (5 div ) were infected with lenti-syn-hTau-mCherry or the empty vector for 7 days, and then, PKA activity (using the phosphor-PKA substrate) and the level of PKAR2alpha (PKA regulatory subunit) in total cell lysis and the nuclear fraction were measured by Western blotting. (d) Overexpressing hTau inhibits proteasome activity. The hippocampal neuron (5 div ) was infected with lenti-syn-hTau-mCherry or the empty vector for 7 days, and the proteasome activity in the cell lysis was measured using a proteasome activity kit. (e, f) Inhibiting proteasome by treating N2a cells with different concentrations of MG132 induces PKAR2alpha elevation with reduced CREB phosphorylation and BDNF expression measured by Western blotting. (g, h) Inhibiting proteasome by treating cultured hippocampal neurons with MG132 (10 muM) for 12 hr induces PKAR2alpha elevation with reduced CREB phosphorylation and BDNF expression measured by Western blotting. Data were expressed as mean +- SEM , * p < .05, ** p < .01 versus Vec, but * p < .05, *

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Overexpressing hTau disrupts PA28gamma-20S proteasome complex formation and downregulating PKAR2alpha or upregulating PA28gamma attenuates hTau-induced PKA/CREB inhibition. (a) Overexpressing hTau increases association of PKAR2alpha with PA28gamma. The cultured hippocampal neurons infected with lenti-syn-hTau-mCherry virus for 7 days, and PA28gamma was co-immunoprecipitated by anti-PA28gamma antibody and detected by Western blotting using anti-PKAR2alpha and anti-PA28gamma antibody. For all co-immunoprecipitations, results are received from three independent experiments. (b) Overexpressing hTau decreases association of PKAR2alpha with 20S proteasome subunit. PKAR2alpha was co-immunoprecipitated using anti-PKAR2alpha antibody and detected by Western blotting using anti-20S proteasome subunit and anti-PKAR2alpha antibody. (c) Overexpressing hTau decreases association of PA28gamma with 20S proteasome subunit. 20S proteasome subunit was co-immunoprecipitated using anti-20S protease subunit and detected by Western blotting using anti-PA28gamma antibody and anti-20S proteasome subunit. (d) Tau dose not bind to PA28gamma. The cultured hippocampal neuron extract was co-immunoprecipitated using anti-PA28gamma, and tau was not detected in the precipitates by Tau5. (e, f) Downregulating PKAR2alpha by shRNA attenuates hTau-induced CREB dephosphorylation. The primary cultured hippocampal neurons ( 5 div ) were co-infected with hTau and shPKAR2alpha lentivirus. After 7 days, the

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