The protein levels of α-SMA, COL1A1, Smad2, p-Smad2, PED7A, PED4A, PKA, p-PKA, total Ras-proximate-1 (Rap1), Guanosine-5′-triphosphate (GTP)-Rap1, and exchange protein directly activated by cAMP 1 (Epac1), cAMP-response element-binding protein (CREB) and p-CREB were examined by immunoblotting following the methods described before (Liu et al., 2018 (link)) using the antibodies listed below: anti-α-SMA (ab5694, Abcam), anti-COL1A1 (ab34710, Abcam), anti-Smad2 (ab40855, Abcam), anti-p-Smad2 (ab53100, Abcam), anti-PDE4A (ab14607, Abcam), anti-PDE4B (ab170939, Abcam), anti-PDE4C (ab170939, Abcam), anti-PDE4D (ab171750, Abcam), anti-PKA (BS-0520R, Woburn, MA, United States), p-PKA (ab75991, Abcam), anti-GTP-Rap1 (ab32373, Abcam), anti-Rap1 (ab14404, Abcam), anti-Epac1 (ab109415, Abcam), anti-CREB (ab31387, Abcam), anti-p-CREB (ab32096, Abcam), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam) and then with HRP-conjugated secondary antibody. Enhanced chemilumescent (ECL) substrates (Millipore, MA, United States) were used for signals visualization using GAPDH as an endogenous protein for normalization.
Ab170939
Manufactured by Abcam
Sourced in United States
Ab170939 is a lab equipment product. It is designed for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab171750, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab32096, by Abcam (1 mentions)
Ab14404, by Abcam (1 mentions)
Ab32373, by Abcam (1 mentions)
Ab14607, by Abcam (1 mentions)
Ab109415, by Abcam (1 mentions)
Ab75991, by Abcam (1 mentions)
Ab40855, by Abcam (1 mentions)
Ab31387, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab53100, by Abcam (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab5694, by Abcam (1 mentions)
3 protocols using ab170939
1
Protein Expression Analysis of Fibrosis Markers
2
Cardiac Protein Signaling Pathway Profiling
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CMs were homogenized in ice-cold RIPA buffer (50 mM Tris pH 7.5, 0.1 M NaCl, 0.1% SDS, 1% NP-40, 1 mM EGTA, 1% Triton X-100, 0.05% sodium deoxycholate, phosphatase, and protease inhibitor cocktails), and protein concentrations were assessed via standard BC assay. A total of 20–30 µg of protein was separated by SDS-PAGE and transferred onto PVDF membrane using standard protocols. Membranes were blotted for total PLN (Abcam, 124174), phosphorylation Ser16 PLN (Badrilla, A010-12), SERCA2a (Enzo, ALX-804-088-RI00), RYR (Sigma, B118272), AKAP7 (Proteintech, 12591-1-AP), PKA RIIα subunit (BD Bioscience, 612242), PDE2A (Fabgennix, PPD2A-101AP), PDE3A (kind gift from Chen Yan), PDE4B (Abcam, ab170939), PDE4D (Abcam, ab171750), and calsequestrin (Thermo Fisher Scientific, PA1-913S). For co-immunoprecipitation (Co-IP) experiments, 800 µg of protein lysate was incubated with total PLN antibody for 8 h at 4°C, followed by an overnight incubation with the beads that were then washed and subjected to either immunoblot analysis or calorimetric PDE activity assay (Abcam, ab241034) according to manufacturer instructions. For subcellular fractionation, heart lysates were applied to sucrose gradient to isolate plasma membrane and sarcolemmal CM fractions exactly as previously described.11 (link)
3
Protein Extraction and Western Blot Analysis
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Cells were homogenized in lysis buffer containing: 1 mmol/L EGTA, 10 mmol/L HEPES, 150 mmol/L NaCl, 300 mmol/L sucrose, 1% Triton-X, and phosphatase and protease inhibitors (Roche, Welwyn Garden City, UK). Protein concentration was determined using Pierce BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Protein lysates were mixed with 4 × SDS sample buffer containing: 200 mmol/L Tris-HCl, 8% SDS, 40% Glycerol, 0.4% Bromophenol blue, and 40 mmol/L DTT, and boiled at 95 °C for 5 min. Proteins (20 µg of total protein per lane) were size separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. For immunodetection primary GAPDH (5G4, dilution 1:160,000, Bio Trend, Koln, Germany), PDE1C (generous gift from C. Yan, dilution 1:1000), PDE2A (PD2A-101AP, dilution 1:500 Fabgennix, Frisco, TX, USA), PDE3A (generous gift from C. Yan, dilution 1:1000), PDE4B (ab170939, dilution 1:2500, Abcam, Cambridge, UK), PDE4D (ab171750, dilution 1:2500, Abcam, Cambridge, UK), and PDE5A (generous gift from S. Rybalkin, dilution 1:100) were used.
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