Immunoblot analysis was performed as previously described [18 (link)]. Total cell lysates were prepared and subjected to immunoblot analysis using the following antibodies: anti-ERK1/2 (1:1000), anti-phospho-ERK1/2 (1:1000), anti-MAPK/ERK kinase 1/2 (MEK1/2) (1:1000), anti-phospho-MEK1/2 (1:1000), anti-phospho-Raf-1 (Ser-338) (1:1000), anti-EGFR (1:1000), anti-phospho-EGFR (Tyr-1068) (1:1000) (Cell Signaling Technology Inc., Beverly, MA, USA), anti-Raf-1 (1:1000) (BD Transduction Laboratories, BD Biosciences Inc., San Jose, CA, USA), or anti-involucrin (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The luminescent signals were analyzed using an LAS-4000 image analyzer (Fuji Film Co., Tokyo, Japan). Immunoblot band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).
Anti involucrin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-involucrin is a laboratory reagent used in immunohistochemistry and Western blotting applications. It is a primary antibody that specifically binds to the involucrin protein, which is a marker for terminal differentiation of epithelial cells. The core function of anti-involucrin is to enable the detection and analysis of involucrin expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor, by Roche (2 mentions)
Anti mnsod, by Merck Group (2 mentions)
Anti sod1, by Santa Cruz Biotechnology (2 mentions)
Anti ck10, by Santa Cruz Biotechnology (2 mentions)
Ecl reagent, by Cytiva (2 mentions)
X ray film, by Kodak (2 mentions)
Anti phospho egfr tyr1068, by Cell Signaling Technology (1 mentions)
Las 4000 image analyzer, by Fujifilm (1 mentions)
Anti phospho mek1 2, by Cell Signaling Technology (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti phospho raf 1 ser 338, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Anti bap31, by Abcam (1 mentions)
Anti au1, by Fortrea (1 mentions)
10 protocols using anti involucrin
1
Immunoblot Analysis of MAPK Signaling
2
Quantitative Western Blot Analysis
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Western blot assays were performed on cell pellet lysates in radioimmunoprecipitation assay buffer with protease inhibitors (Roche, Basel, Switzerland), as previously described13 (link). The antibodies used were anti-involucrin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Western blots were scanned and band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
3
Protein Expression Analysis of Cell Lysates
4
Skin Barrier Disruption and Allergic Inflammation
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The SRMs 1648a and 1649b were purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA) and dispersed in distilled water. SRM 1648a mainly contains heavy metals, whereas SRM 1649b includes polycyclic aromatic hydrocarbons, polychlorinated biphenyl congeners, and pesticides3 (link). Recombinant human interleukin (IL)-4 and IL-13 were purchased from PeproTech (Rocky Hill, NJ, USA). 4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (OXA) was purchased from Sigma-Aldrich (St Louis, MO, USA). OXA was dissolved in vehicle (acetone: olive oil=4:1 mixture). For western blot analysis, anti-occludin, anti-claudin-1, anti-involucrin, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-loricrin and anti-filaggrin were purchased from LifeSpan Biosciences (Seattle, WA, USA). Anti-E-cadherin and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ZO-1 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). A mouse immunoglobulin E (IgE) ELISA kit was purchased from Thermo Fisher Scientific.
5
Keratinocyte Differentiation and Antioxidant Response
6
Western Blot Analysis of Keratinocytes
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The antibodies used in this study were as follows: anti-involucrin and anti-GAPDH (Santa Cruz, Santa Cruz, CA); anti-ATR, anti-p-ATR, anti-ATM, anti-p-p62, anti-GATA4, and Autophagy Antibody Sampler kit, including ATG5, ATG7, ATG16L1, and LC3 (Cell Signaling, Danvers, MA); anti-p62 (American Research Products, Waltham, MA); and anti-p53 (Sigma, St. Louis, MO). Cell lysates were extracted and assayed by Western blot analysis as previously described (43 (link)). Briefly, keratinocytes were separated from J2 feeders by Versene (phosphate-buffered saline [PBS] containing 0.5 mM EDTA) treatment and then lysed in radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min. The samples were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane Immobilon-FL (Millipore, Burlington, MA). The membranes were developed using ECL prime or ECL reagents (Amersham, Pittsburgh, PA), and chemiluminescence signals were visualized using Eastman Kodak X-ray films.
7
Immunoblotting of Keratinocyte Signaling Pathways
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The inhibitors used in this study were as follows: 100 nM UCN01 and 5 µM VE822 (Selleckchem, Houston, TX); 10 µM pimozide (Sigma-Aldrich, St. Louis, MO); and 5 µM CHK2i (Calbiochem, San Diego, CA). The antibodies used in this study were as follows: antiinvolucrin, anti-TopBP1, and anti-GAPDH (Santa Cruz, Santa Cruz, CA) and anti-STAT-5, anti-CHK2, anti-ATM, anti-p-CHK2 (Thr68), anti-p-ATM (Ser1981), anti-CHK1, anti-ATR, anti-p-CHK1 (Ser296), anti-p-CHK1 (Ser345), and anti-p-ATR (Cell Signaling, Inc., Danvers, MA). For Western blot analysis, cell lysates were processed as previously described (36 (link)). Briefly, keratinocytes were first isolated from J2 feeders by treatment with Versene (phosphate-buffered saline [PBS] containing 0.5 mM EDTA) and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer on ice for 30 min. The protein samples were separated on SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were finally developed using Enhanced Chemiluminescence (ECL) Prime or ECL reagents (Amersham, Pittsburgh, PA). Chemiluminescence signals were detected using Eastman Kodak X-ray films.
8
Immunohistochemical Analysis of Epidermal Markers
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All chemical reagents were obtained from Sigma-Aldrich. The antibodies were as follows: anti-loricrin (Abcam, ab24722), working dilution for immunohistochemistry (IHC) 1/1000; anti-involucrin (Santa Cruz, sc-15230), working dilution for IHC 1/1000; anti-cytokeratin 5 (Abcam, ab24647), working dilution for IHC 1/1000; antidesmoglein 1 (Santa Cruz, sc-20114), working dilution for IHC 1/300; anti-desmocollin 1 (Santa Cruz, sc18115), working dilution for IHC 1/250; anti-corneodesmosin (CusAb, CSBPA0051241A01HU), working dilution for IHC 1/500.
9
Immunohistochemical Profiling of Human and Animal Tissues
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Hematoxylin and eosin (H&E) staining and immunohistochemical staining of normal human skin, normal horse skin, breast cancer tissue, and insert’s membranes were performed for Filaggrin, Involucrin, and CK-14. Five-micron sections from formalin-fixed paraffin-embedded tissues were deparaffinized with xylenes and rehydrated through a graded alcohol series. Heat-induced epitope retrieval (HIER) was performed by immersing the tissue sections at 98 °C for 20 min in Tris/EDTA (pH 9.0). Immunohistochemical staining was performed using a horseradish peroxidase-labeled polymer from Dako K4001 according to the manufacturer’s instructions. Briefly, slides were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 min each, and exposed to primary antibodies for 1 h at room temperature. Slides were exposed to the appropriate HRP-labeled polymer for 30 min and DAB chromagen (Dako) for 5 min. Slides were counterstained with hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Consecutive sections with the primary antibody omitted were used as negative controls.
Following antibodies were used: anti-cytokeratin (CK) 14 (Abcam no. ab7800, 1:600,1:5000
Following antibodies were used: anti-cytokeratin (CK) 14 (Abcam no. ab7800, 1:600,
10
Evaluation of Epidermal Keratinocyte Responses
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Human epidermal keratinocytes from the third passage were plated into 96-well plate and after reaching 60% confluency they were treated with 10 -7 M of either 1,25(OH) 2 D 3 , tachysterol 3 , 20(OH)T 3 or 25(OH)T 3 versus ethanol control for 24 h. Cells were fixed and stained with anti-catalase (Santa Cruz Biotechnology), anti involucrin (Santa Cruz Biotechnology) conjugated with Alexa Fluor 488, anti-CK10 (Santa Cruz Biotechnology), anti Mn-SOD (Millipore Corp) or anti-SOD1 (Santa Cruz Biotechnology), at dilutions of 1:100, 1:300, 1:100, 1:50 and 1:50, respectively, as described previously (22, 23, 48, 57) . For secondary antibodies, anti-goat (for CAT and SOD1), anti-mouse (for CK10) both with Alexa Fluor 488, or anti-rabbit with Alexa Fluor 660 (for MnSOD) were used, all at a dilution of 1:200. Images were taken at 10 x magnification using Cytation 5 reader. Nuclei were stained with blue DAPI. Fluorescence intensity was measured using the Cytation 5 reader and calculated using ImageJ software (National Institutes of Health).
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