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PP-1α is a protein phosphatase enzyme that plays a crucial role in the regulation of cellular processes. It functions by dephosphorylating serine and threonine residues on target proteins, thereby modulating their activity and downstream signaling pathways. The core function of PP-1α is to maintain cellular homeostasis and control various cellular processes such as cell cycle progression, metabolism, and gene expression.

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Lab products found in correlation

Calsequestrin, by Dianova (1 mentions) A3682, by Merck Group (1 mentions) Pp2ac, by Merck Group (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Caveolin 3, by BD (1 mentions) Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Troponin 1, by Cell Signaling Technology (1 mentions) Pser 23 24 troponin 1, by Cell Signaling Technology (1 mentions) Gapdh, by Biosynth (1 mentions)

2 protocols using pp 1α

1

Cardiac Protein Phosphorylation Analysis

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After incubation with H2O2, NRCMs were lysed with ice-cold GST-fish buffer (10% glycerol, 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-4, 4 mM MgCl2, 1% IGEPAL CA-630, complete protease inhibitor), cell debris was removed at 13,000 g (10 min, 4 °C). For non-reducing 12–15% acrylamide SDS-PAGE, the cells were lysed in the presence of 10 mM maleimide but without DTT and heat. Gels were blotted onto a nitrocellulose membrane, blocked with 1x Roti-Block (Roth) for 1 h, washed with TBST (Tris-buffered saline, 0.1% Tween 20) and incubated overnight at 4 °C with a primary antibody: calsequestrin (1:1000, Source: rabbit, Dianova, ABR-01164), PP-1α (1:200, Source: goat, Santa-Cruz, sc-6104), PP-2Ac (1:1000, Source: rabbit, Millipore, 07–324), PKA-RI (1:200, Source: mouse, BD Transduction laboratories, 610166), PLB-pSer16 (1:5000, Source: rabbit, Badrilla Ltd., A010–12), cMyBPC-pSer282 (1:5000, Source: rabbit, Enzo Life Science, ALX-215–057-R050), and I-1-pThr35 (1:1000, Source: rabbit, Cell signalling, #2302). The membrane was washed three times with TBST for 10 min each and incubated for 1 h with the secondary antibody: HRP-coupled antibodies (mouse: Sigma-Aldrich, A3682; rabbit: Sigma-Aldrich, A0545; goat: Santa-Cruz, sc-2020).
Singh S., Lämmle S., Giese H., Kämmerer S., Meyer-Roxlau S., Alfar E.A., Dihazi H., Guan K., El-Armouche A, & Richter F. (2018). The reduced activity of PP-1α under redox stress condition is a consequence of GSH-mediated transient disulfide formation. Scientific Reports, 8, 17711.
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2

Subcellular Fractionation and Protein Analysis

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Subcellular protein fractions were prepared as described previously [32 (link)]. In brief, cells were washed and collected in lysis buffer containing 50 mM Tris-HCl pH 7.4, 5 mM EGTA, 2 mM EDTA, 5 mM DTT, 0.05% digitonin, and a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78440). Cell lysates were centrifuged at 14,000 x g for 15 min, and the supernatant was collected as a cytosolic fraction. The pellet was resuspended in lysis buffer containing 1% Triton x-100 for 10 min and centrifuged for 15 min at 14,000 x g to collect the supernatant as the membrane fraction. The subsequent triton-insoluble pellet contained the myofilament fraction. This insoluble pellet was resuspended with PBS buffer containing 0.5 M NaCl for 20 min on ice and centrifuged to collect supernatant as the final myofilament protein fraction. Protein samples from each fraction were quantified with a Bradford assay (Bio-Rad) and subjected to 10% SDS-PAGE for Western blot detection of PP1α (Santa Cruz Biotechnology, sc-6104), PP1β (Millipore, 07-1217), PP1γ (Santa Cruz Biotechnology, sc-6108), GAPDH (Fitzgerald, 10-1500), Troponin I (Cell Signaling Technology, 4002), pSer 23/24-Troponin I (Cell Signaling Technology, 4004), Caveolin-3 (BD Biosciences, 610421), I-1 (Abcam, ab40877), and I-2 (R&D Systems, AF4719).
Liu R., Correll R.N., Davis J., Vagnozzi R.J., York A.J., Sargent M.A., Nairn A.C, & Molkentin J.D. (2015). Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations. Journal of molecular and cellular cardiology, 87, 204-213.
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