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.
Anti pkcα
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-PKCα is a primary antibody that recognizes the protein kinase C alpha (PKCα) enzyme. PKCα is a serine/threonine-specific protein kinase that plays a crucial role in various cellular processes. The Anti-PKCα product provides a specific tool for the detection and study of PKCα in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pkcδ, by Cell Signaling Technology (3 mentions)
Anti p pkc α, by Cell Signaling Technology (2 mentions)
Odyssey fc system, by LI COR (2 mentions)
Anti egfr e746 a750del specific, by Cell Signaling Technology (2 mentions)
Mach 3 rabbit hrp, by Biocare Medical (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Anti p pkc δ, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
Anti total erk1 2, by Cell Signaling Technology (1 mentions)
Anti total p38, by Cell Signaling Technology (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Anti actin, by Bio-Rad (1 mentions)
Anti pkcζ, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Fluorchem fc2, by Bio-Techne (1 mentions)
8 protocols using anti pkcα
1
Western Blot Analysis of Vascular Proteins
2
Western Blot Analysis of Signaling Proteins
3
Peritoneum Protein Expression Analysis
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The peritoneum samples of the piglets were collected, dissociated by RIPA lysis buffer supplemented with protease inhibitor mixture and centrifuged at 12,000 g for 15 min at 4 °C. The total protein was measured with BCA protein extraction kit (Beyotime). Subsequently, samples with the same amount of protein (80 μg) were fractionated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% skimmed milk for 3 h, the PVDF membranes were incubated with special primary antibody (containing 5% BSA TBS-T solution, 1:1000 dilution, rabbit anti-β-actin, anti-PKC-α, anti-p-PKC-α, Cell Signaling, Danvers, MA, USA; anti-MLCK, anti-MLC-2 and anti-p-MLC-2, Abcam, Shanghai, China) at 4 °C overnight, and then incubated with the corresponding HRP labeled secondary antibodies (1:4000 dilution) at 37 °C for 3 h. Protein level was determined using the enhanced chemiluminescent (ECL) reagent (Beyotime) and the images were captured with a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Quantitative analysis was carried out using FluorChem FC2 (Alpha Innotech, San Leandro, CA, USA). The β-actin was used as the inner loading control. Gray value was analyzed and the relative expression level of protein was obtained.
4
Immunohistochemical Analysis of Protein Targets
5
Western Blot Analysis of PKC Isoforms
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Western blots were done essentially as previously described66 (link). Briefly, A549 cells were harvested in lysis buffer containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, and 2% β-mercaptoethanol. Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA). After blocking with 5% milk or 5% BSA in Tris-buffered saline/0.1% Tween for 1 h, membranes were incubated overnight with the following primary antibodies: anti-PKCα, anti-PKCδ, anti-PKCε (all from Cell Signaling, 1:1,000 dilution, Catalog #2056, #2058, and #2083, respectively), vinculin (Sigma-Aldrich, 1:5,000, catalog #V9131) or β-actin (Sigma-Aldrich, 1:50,000 dilution, catalog #A5441). Membranes were then incubated for 1 h with either anti-mouse (1:1,000 dilution) or anti-rabbit (1:3,000 dilution) secondary antibodies conjugated to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA). Bands were visualized and subjected to densitometric analysis using an Odyssey Fc system (LI-COR Biotechnology, Lincoln, NE).
6
Comprehensive Anticancer Pathway Analysis
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FAP antibody was purchased from Cell Signaling (catalog 66562). DOXO (catalog D1515), PTX (catalog T7402), and CCR2 antagonist (catalog 227016) were purchased from Sigma-Aldrich. Navitoclax (ABT-263) (catalog S1001) was purchased from Selleckchem. Recombinant IL-6 protein (catalog 7270-IL), recombinant WNT5A protein were acquired from R&D System (catalog 645-WN). Tocilizumab was acquired from Novus Biologicals (catalog NBP2-75193). Puromycin was acquired from Thermo Fisher Scientific (catalog A11138). Cell Signaling Technology antibodies contributed: anti–PKC-α (catalog 59754S), anti–p-PKC-α/β II (catalog 9375S), anti–p-PLC-γ1 (catalog 8713S), anti–PLC-γ1 XP (catalog 5690S), anti–p-RAC1/cdc42 (Ser71) (catalog 2461), anti-RAC1/2/3 (catalog 2465), anti-WNT5A/B (catalog MA5-15502), and anti–cleaved caspase-3 (catalog 9661S). Mouse monoclonal β-actin–HRP (catalog 47778, Santa Cruz Biotechnology) was used as loading control. Anti-Ki67 was acquired by BD Horizon (catalog 565929).
7
Western Blot Analysis of Signaling Pathways
8
Immunohistochemical Analysis of Protein Targets
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Human tissue arrays were obtained from Folio (Catalogue number: FOLIO-800). Deparaffinization of tissue slides was performed at 60°C for 1 hr. After 10 min of cooling, tissues were hydrated in two separate baths of xylene for 5 minute each, followed by a bath in 2% hydrogen peroxide/methanol solution for 30 minutes and finally incubated in 3 different concentrations of ethanol solutions (100%, 95%, 70%). After neutralizing in water, antigen retrieval was performed in citrate buffer (pH 6) at 100–120°C for 10 minutes. Sections were cooled at 4°C for 30 min and subsequently blocked using 5% BSA/Tris buffer solution/ 0.1% Tween (TBST) for 30 min at room temperature. Blocked tissues were incubated with unconjugated primary antibody overnight: anti-EGFR (E746-A750del Specific) or anti-PKCα (Cell Signaling), both diluted 1:50 in block solution. Tissues were washed for 5 min 3x with TBST and incubated with conjugated secondary antibody (Biocare MACH 3 Rabbit HRP). Sections were then washed and counterstained with nuclear stain and mounted in Vectashield mounting medium (Vector Labs). Microscopy was performed using a Zeiss microscope. Optical density (OD) for PKCα staining were calculated by ImageJ software version 2 (https://imagej.nih.gov/ij/ ).
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