Anti pka c α

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Anti-PKA C-α is a primary antibody that recognizes the catalytic subunit alpha of Protein Kinase A (PKA). PKA is a key regulator of cellular processes and plays a crucial role in signal transduction pathways.

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PKA C-α (21 citations) Anti-PKA (9 citations) Rabbit anti-PKA C-α (2 citations)

14 protocols using anti pka c α

Nonanal-Induced Protein Expression Analysis

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Western blotting was performed to determine the protein levels. DPCs were seeded in a 60-mm dish at a density of 200,000 cells, treated with either the vehicle or nonanal, and harvested at various time points. The cells were washed with HEPES buffer and lysed in a PRO-PREP protein extraction buffer (iNtRON; Seoul, Korea). Overall, 20 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Whatman; Kent, UK). Subsequently, the membrane was blocked with 5% bovine serum albumin (BSA; MP biomedicals; Irvine, CA, USA) in tris-buffered saline/Tween-20 (TBST) for 1 h and then incubated with a 1:1000-diluted anti-PKA Cα, anti-β-catenin, or anti-GAPDH antibodies (Cell Signaling; Herts, UK) overnight at 4 °C. The membrane was further incubated with a peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology; Santa Cruz, CA, USA) for 1 h at 20 °C. Antibody binding was detected with electrochemiluminescence detection reagent (ECL; BioRad) and visualized using Ez-capture (ATTO; Tokyo, Japan). The protein levels were normalized to those of the loading control (GAPDH).

Park S., Kang W., Choi D., Son B, & Park T. (2020). Nonanal Stimulates Growth Factors via Cyclic Adenosine Monophosphate (cAMP) Signaling in Human Hair Follicle Dermal Papilla Cells. International Journal of Molecular Sciences, 21(21), 8054.

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Insulin Signaling Pathway Analysis

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INS 832/13 cells were grown to 100% confluence in 6-well plates. Cells were treated with wortmannin (100 nM) or equivolume DMSO (0.1%) in KRB at the beginning of the 2.8 mM glucose 2-hr pre-incubation at 37 °C; these compounds were present for the duration of the experiment. Cells were treated with insulin (200 nM) or forskolin (1 μM) for 15 min in 16.7 mM KRB. Cells were then washed once with PBS, and then lysed using a standard RIPA lysis buffer supplemented with phosphodiesterase and protease inhibitor cocktails (Millipore, Etobicoke, ON).
Cell lysates were subjected to SDS-PAGE on Mini-PROTEAN TGX Stain-Free gels (Bio-Rad, Mississauga, ON) transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and probed with primary antibodies: anti-phospho-(Ser/Thr) AKT substrate; #9611, anti-phospho-(Ser/Thr) PKA substrate; #9621, anti-AKT; #9272, anti-PKA C-α; #4782 (Cell Signaling Technology, Beverly, MA) and anti-β-actin; sc-4778 (Santa Cruz Biotechnology). Detection was with peroxidase-conjugated secondary anti-rabbit (NA934V) and anti-mouse antibodies (NA931V) (GE Healthcare), and visualization by chemiluminescence with ECL-Plus (GE Healthcare). Images were acquired using a ChemiDoc MP System (Bio-Rad) and analyzed using Image Lab Software 5.2.1 (Bio-Rad).

Kolic J., Manning Fox J.E., Chepurny O.G., Spigelman A.F., Ferdaoussi M., Schwede F., Holz G.G, & MacDonald P.E. (2016). PI3 kinases p110α and PI3K-C2β negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets. Molecular Metabolism, 5(7), 459-471.

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Signaling Pathway Modulation in Thyroid Cells

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Cells were starved and pretreated with U0126 (5 μM), SB203580 (5 μM), H89 (10 μM), SP600125 (5 μM) or wortmannin (0.5 μM) for 1 h and then stimulated with 10 nM TSH for 60 min. Cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA and 1% Triton-X 100) with a protease inhibitor cocktail for 30 min at 4°C. Lysates were quantified using a BCA protein assay kit (Pierce) according to the manufacturer's instructions. Proteins in the lysate were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). After blocking with 5% skimmed milk, the membranes were incubated with anti-PAK4 (1:1000), anti-p-PAK4 (1:1000), anti-PKA Cα(1:1000) (Cell Signaling Technology, Beverly, MA, USA), anti-TSHR (1:1000) (Santa Cruz), cAMP (1:1000) (Sigma) and anti-GAPDH (1:2000) (GenScript Corporation, Nanjing, China) antibodies. U0126, SB203580, H89, SP600125, wortmannin, and forskolin were purchased from BioTime Technology.

Xie X., Shi X., Guan H., Guo Q., Fan C., Dong W., Wang G., Li F., Shan Z., Cao L, & Teng W. (2017). P21-activated kinase 4 involves TSH induced papillary thyroid cancer cell proliferation. Oncotarget, 8(15), 24882-24891.

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Immunofluorescence Staining of Cellular Proteins

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Following antibodies were used: anti-GAPDH (HRP-60004, Proteintech), anti-6∗His (ab18184, Abcam), anti-GFP (11814460001, Roche), anti-Myc (2278S, Cell Signaling Technology), anti-ACBD3 (H00064746-B01P, Abnova), anti-P-PKA-substrate(9621S, Cell Signaling Technology), anti-Golgin97 (13192, Cell Signaling Technology), anti-mCherry (ab167453, Abcam), anti-PKA-Cα (#4782, Cell Signaling Technology), anti-PKA-RIIα (612242, BD), anti-γ-COP (sc-393615, Santa Cruz), anti-GM130 (610822, BD Bioscience), anti-SNAP tag (P9310S, NEB), anti-Clathrin HC (ab21679, Abcam), anti-ARF1 (NB300-505, Novus), anti-β-Actin (3700S, Cell Signaling), and anti-α-Tubulin (3873S, Cell Signaling). Anti-Rabbit Alexa Fluor 488 (A21441), Alexa Fluor 568 (A10042), Alexa Fluor 647 (A21245), and anti-Mouse Alexa Fluor 488 (A21200), Alexa Fluor 568 (A10037), and Alexa Fluor 647 (A21236) for immunofluorescence staining were obtained from ThermoFisher. D/D Solubilizer (635054) was purchased from Clontech. Forskolin (F6886) was purchased from Sigma.

Jia J., Tang S., Yue X., Jing S., Zhu L., Tan C., Gao J., Du Y., Lee I, & Qian Y. (2023). An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi. The Journal of Biological Chemistry, 299(5), 104696.

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Immunoblotting of B Cell Signaling

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B cells cultured under various conditions were harvested and cell lysates were prepared in lysis buffer. The lysate was resolved on a 10% reducing SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% dried skimmed milk, the membrane was incubated with antibodies, followed by probing with HRP-conjugated anti-rabbit or -mouse antibody (Sigma). The bands were detected by chemiluminescence with ECL detection reagents (Life Technologies). Antibody to Phospho-STAT6 (Tyr641, 56,554), anti-STAT6 (5,397), anti-p-PKA C(4,781), anti-PKA C-α (5,842), anti-p-CREB (9,198), anti-CREB (9,197), PI3 Kinase p110 δ 34,050, anti-p-Akt (Ser473, 4,060), anti-Akt (4,685), anti-p-FoxO1 (Ser256, 9,461), anti-FoxO1 (2,880), anti-p-FoxO3a (Ser253, 13,129), anti-FoxO3a (12,829), anti-PPARγ (2,443) were from Cell Signaling Technology (Danvers, MA, USA). Anti-ubiquitin (PTM-1106) was from PTM BIO (Beijing, China). Anti-β-Actin (66009-1-Ig) was from Proteintech.

Wu J., Wang Y., Zhou Y., Wang Y., Sun X., Zhao Y., Guan Y., Zhang Y, & Wang W. (2020). PPARγ as an E3 Ubiquitin-Ligase Impedes Phosphate-Stat6 Stability and Promotes Prostaglandins E2-Mediated Inhibition of IgE Production in Asthma. Frontiers in Immunology, 11, 1224.

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Co-Immunoprecipitation of p75NTR Signaling Complex

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Co-IP was performed as described previously (Le Moan et al., 2011 (link)). Cell lysates were prepared in 1% NP-40, 200 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl, pH 8.0. Rabbit anti-p75^NTR antibody 9992 (kind gift of Moses Chao), rabbit anti-PKACα (Cell Signaling), and rabbit anti-perilipin (Cell Signaling) were used for IP, and immunoblots were performed with anti-PKACα, anti-PKARIIα, anti-PKARIIβ, anti-PDE4A, anti-phospho PKA substrate, and anti-p75^NTR. Additionally, rabbit anti-HA and mouse anti-Myc were used for IP, and immunoblots were performed with anti-GFP, anti-HA, and anti-Myc.

Baeza-Raja B., Sachs B.D., Li P., Christian F., Vagena E., Davalos D., Le Moan N., Ryu J.K., Sikorski S.L., Chan J.P., Scadeng M., Taylor S.S., Houslay M.D., Baillie G.S., Saltiel A.R., Olefsky J.M, & Akassoglou K. (2015). p75 neurotrophin receptor regulates energy balance in obesity. Cell reports, 14(2), 255-268.

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Immunoblotting Analysis of Kinase Signaling

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For direct analysis, cells were lysed using RIPA buffer whereas for kinobead analysis, elutions from kinobeads were mixed with an equal volume of 2× loading buffer prior to SDS-PAGE. Immunoblotting was performed using anti-phospho-SYK (Y525/6), anti-SYK, anti-phospho-BTK (Y223), anti-BTK, anti-phospho-GAB1 (Y627), anti-GAB1, anti-phospho-TBK1, anti-TBK1, anti-phospho-PKAC, anti-PKACα, anti-phospho-ERK1/2 (Y202/T204), anti-ERK, anti-phospho-AKT (S473), and anti-AKT, (all Cell Signaling Technologies). Anti-actin (Sigma) and anti-HSC70 (Insight Biotechnology) were used as loading controls. Bound primary antibodies were detected using species specific fluorophore-conjugated secondary antibodies (LI-COR Biosciences) and imaged using a LI-COR Odyssey CLx instrument.

Linley A.J., Karydis L.I., Mondru A.K., D'Avola A., Al Shmrany H., Cicconi S., Griffin R., Forconi F., Pettitt A.R., Kalakonda N., Rawstron A.C., Hillmen P., Steele A.J., MacEwan D.J., Packham G., Prior I.A, & Slupsky J.R. (2021). Kinobead Profiling Reveals Reprogramming of BCR Signaling in Response to Therapy within Primary CLL Cells. Clinical Cancer Research, 27(20), 5647-5659.

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Western Blot Analysis of Signaling Proteins

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Hs68 cells were lysed in a protein extraction buffer (iNtRON; Seoul, Korea). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose membrane (Whatman; Kent, UK). The membrane was blocked for 1 h in 5% bovine serum albumin (BSA; MP Biomedicals; Auckland, New Zealand) in Tris-buffered saline/Tween-20 (TBST), and probed with primary antibodies at 4 °C overnight. The primary antibodies used were anti-PKA Cα (Cell Signaling; Herts, UK; 1:1000), anti-GAPDH (Cell Signaling; 1:5000), anti-pCREB (Cell Signaling; 1:1000), and anti-CREB (Cell Signaling; 1:1000). Then, the membrane was incubated with peroxidase-conjugated secondary antibody (Sigma) at 20 °C for 1 h. Protein levels were detected using an electrochemiluminescence detection reagent (ECL, BioRad) using Ez-capture (ATTO; Tokyo, Japan). Quantification was performed using the ImageJ program (National Institutes of Health; Bethesda, MD, USA).

Son B., Kang W., Park S., Choi D, & Park T. (2021). Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells). International Journal of Molecular Sciences, 22(17), 9273.

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Protein Expression Analysis of Pancreatic Cells

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Cultured islets or Min6 cells were washed in PBS and lysed. PVDF membranes were incubated with anti-TCF7L2 (#2565), anti-actin (#4967), anti-p-AKT (Serine473, #9271), anti-AKT (#9272), anti-p-GSK3β (Ser9 #9336), anti-PARP (#9542), anti-GAPDH (#2118), anti-c-casp3 (#9661), anti-stat3 (#9132), anti-p-stat3 (Tyr705, #9131), anti-PKA C-α ( #5842; all from Cell Signaling, Danvers, MA, USA), anti-β-catenin (ab6302), anti-GLP-1R (ab39072), anti-p-Jak2 (ab68268; all from Abcam), followed by incubation with horseradish-peroxidase-linked IgG peroxidase. The bands were visualized and densities of the bands were analyzed using Tanon ChemImaging Systems (Nanjing, China).

Yao D.D., Yang L., Wang Y., Liu C., Wei Y.J., Jia X.B., Yin W, & Shu L. (2015). Geniposide promotes beta-cell regeneration and survival through regulating β-catenin/TCF7L2 pathway. Cell Death & Disease, 6(5), e1746-.

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Comprehensive Antibody Panel for Cell Analysis

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The following antibodies were used: anti-GATA3 (HG3-31, sc-268), anti-cyclin A (C-19, sc-596), anti-cyclin E (HE12, sc-247), anti-ER (MC-20, sc-542) and anti-PR (H-190, sc-7208), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-pSer308-GATA3 (ab61052), anti-Histone H3 (tri-methyl K27) (ab6002) and anti-Histone H3 (acetyl K9) (ab4441) from Abcam (Cambridge, MA, USA); anti-PR (Ab7), anti-actin (clone ACTN05) and anti-cyclin D1 (RB 9041-P1) from Neomarkers (Freemont, CA, USA); anti-GAPDH (D16H11), anti-phospho-PKA Substrate (RRXS*/T*)(100G7E) and anti-PKA C-α (4782) from Cell Signaling (Beverly, MA, USA); anti-EZH2 (#39933) from Active Motif (Carlsbad, CA,USA); anti-acetyl-Histone H4 (#06-866) from Millipore (Temecula, CA, USA); and anti-β-tubulin from Sigma.

Izzo F., Mercogliano F., Venturutti L., Tkach M., Inurrigarro G., Schillaci R., Cerchietti L., Elizalde P.V, & Proietti C.J. (2014). Progesterone receptor activation downregulates GATA3 by transcriptional repression and increased protein turnover promoting breast tumor growth. Breast Cancer Research : BCR, 16, 491.

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