Both liver tissues and cell lysates were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis before transferring to nitrocellulose membranes. After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: anti-phospho-STAT3, anti-total STAT3, anti-IκBα, anti-PKCδ, and anti-PTEN obtained from Cell Signaling Technology (Danvers, MA); anti-GSK3β from Abcam (Cambridge, UK); and anti-p38, anti-pERK, and anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were washed and incubated with peroxidase-conjugated secondary antibodies (Amersham Bioscience, Amersham, UK).
Anti pkcδ
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-PKCδ is a specific antibody that recognizes the protein kinase C delta (PKCδ) isoform. PKCδ is a member of the protein kinase C family of enzymes involved in various cellular signaling pathways. The Anti-PKCδ antibody can be used to detect and study the expression and distribution of PKCδ in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pkcε, by Cell Signaling Technology (5 mentions)
Anti pkcε, by Santa Cruz Biotechnology (3 mentions)
Anti vimentin, by Cell Signaling Technology (3 mentions)
Anti pkcα, by Cell Signaling Technology (3 mentions)
Anti rhoa, by Cell Signaling Technology (2 mentions)
Anti cdc42, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by R&D Systems (2 mentions)
Anti phospho smad2 3, by Cell Signaling Technology (2 mentions)
Anti phospho mypt1, by Cell Signaling Technology (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti β actin, by BD (2 mentions)
Odyssey fc system, by LI COR (2 mentions)
Anti pkcζ, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti p erk, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti gsk3β, by Abcam (1 mentions)
Anti pten, by Cell Signaling Technology (1 mentions)
Anti total stat3, by Cell Signaling Technology (1 mentions)
14 protocols using anti pkcδ
1
Western Blot Analysis of Signaling Proteins
2
Isolation and Immunoblotting of Mitochondrial and Cytosolic Proteins
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Total proteins isolated from LV tissues were rapidly minced and homogenized in 1 × RIPA ice‐cold lysis buffer (with protease inhibitor). After centrifuging at 800 g for 5 min. at 4°C to remove nuclei, the supernatant was further centrifuged at 12,000 g for 30 min. to obtain the mitochondrial pellets and the cytosolic extracts (supernatant). Equal amount of mitochondrial fractions or cytosolic proteins was separated in 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore). The membranes were immunoblotted with anti‐Drp1Ser616, anti‐caspase9, anti‐PKC‐δ, anti‐PKC‐ε, anti‐GSK‐3β and anti‐GSK‐3βSer 9 (Cell Signaling, Beverly, MA, USA) at 4°C overnight. After washing by 0.1%PBS for three times, the blots were incubated with HRP‐conjugated anti‐IgG for 2 hrs. Immunoreactivities were detected using the enhanced chemiluminescence reaction system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Densitometric analysis was performed using QuantityOne software version 4.5.2 (Bio‐Rad, Hercules, CA, USA). In brief, the density area of each band can be automatically identified and outlined by the software, and then the brightness value for each band was obtained. The ratio of each detected protein to β‐actin represented to their relative protein levels.
Densitometric analysis was performed using QuantityOne software version 4.5.2 (Bio‐Rad, Hercules, CA, USA). In brief, the density area of each band can be automatically identified and outlined by the software, and then the brightness value for each band was obtained. The ratio of each detected protein to β‐actin represented to their relative protein levels.
3
Western Blot Analysis of Key Signaling Proteins
4
Western Blot Analysis of Vascular Proteins
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Protein expressions of PKC-α, PKC-δ, p-PKC-α, p-PKC-δ, MLCK, MLC-2, and p-MLC-2 were detected by Western blotting analysis. Total protein extraction kit (Beyotime, China) and BCA protein assay kit (Sigma, USA) were used to extract the total protein in aorta and measure the protein concentrations. The total proteins were fractionated using 10% SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes, and then blocked for 3 h with 5% skimmed milk. After incubation with special antibodies (diluted at 1:1,000, including 5% BSA TBS-T, rabbit anti-β-actin, anti-PKC-δ, anti-PKC-α, anti-p-PKC-δ, and anti-p-PKC-α, Cell Signaling, USA; anti-MLCK, anti-MLC-2, and anti-p-MLC-2, Abcam, China) at 4°C for 14 h, corresponding HRP labeled secondary antibodies (diluted at 1:4,000) were added and incubated at 37°C for 3 h. Enhanced chemiluminescent (ECL) reagent (Beyotime, China) was added to determine the protein expression levels, and Azure Bio-imaging systems (California, USA) were used to capture the images. FluorChem FC2 AIC system (Alpha, USA) was used for quantitative analysis. The internal control used was β-actin.
5
Western Blot Analysis of Signaling Proteins
6
Western Blot Analysis of EMT Markers
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Western blot analysis (WB) was carried out essentially as previously described in (63 (link)). The detection and quantification were performed with Odyssey Clx System (LI-COR Biosciences) through the Image Studio Software, or by traditional ECL detection. The following antibodies were used: anti-ZEB1-1642 (from Dr. Douglas Darling Lab), anti-ZEB1 (Santa Cruz,# sc-25388), anti-E-cadherin (BD Biosciences,# 610182), anti-vimentin (Cell Signaling, #5741), anti-SNAIL (Cell Signaling, #3879), anti β-catenin (Cell Signaling, #8480), anti ZO-1 (Cell Signaling, #8193), anti-cytokeratin 18 (Cell Signaling, #4548), anti-phospho serine/threonine (Abcam, #ab9337), anti-phospho-substrates antibodies kit Cell Signaling, #9920) anti-GFP (Abcam, #ab290), anti-PKCα (Santa Cruz, # sc-208), anti-PKCδ (Cell Signaling, #2058) anti-PKCε (Santa Cruz,# sc-1681), anti- Phospho-PKCα/β II (Cell Signaling, #9375) anti-α-tubulin (Sigma-Aldrich, #T5168); anti-β-actin (Sigma-Aldrich, #A2228); anti-PCNA (Santa Cruz, #sc56). As secondary antibodies we used anti-rabbit-Alexa-Fluor 594 (Molecular Probes), anti-rabbit HRP (Cell Signaling, #7074), anti-mouse HRP (Cell Signaling, #7076), goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (LI-COR Biosciences).
7
Antibody Procurement and Characterization
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An anti‐GFP antibody (#598) was purchased from MBL, Nagoya, Japan. An anti‐FLAG M2 antibody conjugated with peroxidase was purchased from Sigma (St. Louis, MO, USA). The anti‐β‐actin antibody (N21) and horse radish peroxidase (HRP)‐conjugated anti‐goat IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐Akt (#9272), anti‐phospho‐Akt (Ser473) (#9271), anti‐CDCP1 (#4115), anti‐phospho‐CDCP1 (Tyr734) (#9050), anti‐ERK1/2 (#9102), anti‐phospho‐ERK1/2 (Thr202/Tyr204) (#9101), anti‐PKCδ (#2058), and anti‐phospho‐PKCδ (Tyr311) (#2055) antibodies were purchased from Cell Signaling Technology (Denvers, MA, USA). anti‐CDCP1 antibody for immunoprecipitation was originally developed as described previously.2
8
Quantitative Western Blot Analysis
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The protein level was quantified by Western blot; cells were washed in PBS and lysed with Radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor. Proteins were quantified by a Bicinchoninic Acid method (BCA) Pierce protein assay kit (Thermo Fisher Scientific, USA). Proteins were resolved by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked with 5% nonfat dry milk and probed with primary antibodies anti-beta-actin 1:5000 (Abcam, Cambridge, USA) or anti-PKCδ 1:1000 (Cell Signaling Technology, USA) overnight. The following day, the membranes were incubated with appropriate horseradish peroxidase- (HRP-) conjugated secondary antibodies anti-mouse 1:10000 or anti-rabbit 1:25000, respectively, for 1 h (Abcam, Cambridge, USA). The signals were visualized with an ECL kit (Pierce, Thermo Fisher Scientific, USA) using an X-ray film.
9
Platelet Activation Signaling Pathway
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U46619, 2-MeSADP, ADP, thrombin, serotonin, epinephrine, apyrase (type V), prostaglandin E1 (PGE1), sodium citrate, and acetylsalicylic acid were from Sigma (St. Louis, MO, USA). CRP was obtained from Dr. Richard Farndale at the University of Cambridge. Phycoerythrin-conjugated antibody JON/A and FITC-conjugated anti-P-selectin antibody were from Emfret Analytics (Sterntalerweg, Wurzburg, Germany). Hexapeptide AYPGKF was custom synthesized by Invitrogen (Carlsbad, CA, USA). Fura-2-AM was from Millipore (Temecula, CA, USA). Anti-phospho-Akt (Ser473), anti-phospho-ERK (Thr202/Tyr204), anti-phospho-PKCδ (Tyr311), anti-Akt, anti-PKCδ, and anti-ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-labeled secondary antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were of analytical grade, and deionized water was used throughout the study.
10
Pressure-Overload-Induced Cardiac Signaling
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