Cellular proteins were separated using SDS-PAGE, transferred to nitrocellulose membranes, and incubated with appropriate antibodies overnight at 4℃ with gentle agitation. The blots were incubated with peroxidase-conjugated secondary antibodies for 2 h at room temperature, and the signals were visualized using enhanced chemiluminescence (Intron, Daejeon, Korea). We used primary antibodies against PAR-2, phosphorylated-ERK (p-ERK), filaggrin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and loricrin (Covance Research Products, Denver, PA, USA).
Filaggrin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Filaggrin is a protein that plays a crucial role in the structure and function of the skin's outer layer, the stratum corneum. It is responsible for aggregating and aligning keratin filaments, which contribute to the formation of the skin's protective barrier. Filaggrin is essential for the skin's moisture retention and barrier function.
Automatically generated - may contain errors
Lab products found in correlation
Involucrin, by Santa Cruz Biotechnology (3 mentions)
Loricrin, by GeneTex (2 mentions)
Cytokeratin14, by GeneTex (2 mentions)
Cytokeratin15, by GeneTex (2 mentions)
Cytokeratin10, by GeneTex (2 mentions)
Fluorescently coupled secondary anti rabbit or rat igg, by Cell Signaling Technology (2 mentions)
Streptavidin conjugated cy5, by BioLegend (2 mentions)
Leica dmi6000b fluorescence microscope, by Leica Microsystems (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Involucrin, by Fortrea (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Involucrin, by Proteintech (1 mentions)
Loricrin, by Proteintech (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Cytokeratin 10, by Santa Cruz Biotechnology (1 mentions)
Cytokeratin 14, by Santa Cruz Biotechnology (1 mentions)
Cytokeratin 1, by Santa Cruz Biotechnology (1 mentions)
Krt18, by Santa Cruz Biotechnology (1 mentions)
13 protocols using filaggrin
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Epidermal Proteins
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The cells were lysed in CETi lysis buffer (TransLab) for 30 min, after which the lysate was centrifuged and the supernatant was collected. The total protein concentration of the lysate was evaluated using a BCA protein assay kit (Thermo Scientific) standardized to BSA, as per the manufacturer's instruction. Estimated cell lysates were then resolved by SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with antibodies against filaggrin, phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), involucrin, or loricrin (Proteintech, Rosemont, IL, USA), and subsequently incubated with HRP-conjugated secondary antibodies, including goat anti-rabbit or rabbit anti-mouse antibodies. Finally, the blots were developed using an enhanced chemiluminescence detection system (Thermo Scientific) and visualized by Chemidoc (Bio-Rad).
3
Immunofluorescence Staining of Epidermal Markers
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Cells were fixed in 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized in 0.5% Triton X-100/PBS for 10 minutes. After blocking with 3% BSA/PBS for 1h, cells were incubated with primary antibodies overnight in 4℃. Cells were then washed in PBS, incubated with the secondary antibodies for 1 hour and washed with PBS three times. Hoechst 33258 (Molecular Probes H1398) was used for nuclei staining (10min). The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).
4
Immunostaining of Skin Cryosections
5
Inflammatory Signaling Pathway Modulation
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Millipore Sigma (Billerica, MA, USA). Recombinant human TNF-α and recombinant human IFN-γ were purchased from Bio-Techne Ltd. (Abingdon, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Primary antibodies against p-IKK α/β (cat no. 2697), NF-κB p65 (cat no. 8242), p-Akt (cat no. 9271), and ICAM-1 (cat no. 4915) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against IKK α/β (cat no. 7607), p-IκB-α (cat no. 8404), IκB-α (cat no. 203), Akt1/2/3 (cat no. sc-8312), PARP (cat no. sc-9542), α-tubulin (cat no. sc-8035), Filaggrin (cat no. sc-66192), VCAM-1 (cat no. sc-1504), E-selectin (cat no. sc-5262), and β-actin (cat no. sc-81178) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories, Inc. (West Grove, PA, USA). The histamine ELISA kit was obtained from Enzo life Sciences, Inc. (Farmingdale, NY, USA). The ELISA kits for TNF-α and IL-6 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).
6
Immunoblotting of Cellular Proteins
7
Protein Expression Analysis in HaCaT Cells
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Total cellular protein from HaCaT monolayers was resolved by 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using standard protocols. Primary antibodies against the following antigens were used: filaggrin (goat polyclonal antibody; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and keratin 10 (rabbit monoclonal; Abcam, Cambridge, UK). Densitometric analysis was performed with the VersaDoc Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA); all bands were background-corrected and normalized to keratin 10.
8
Immunohistochemical Analysis of ILVEN, PV, and Controls
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This study collected data from 8 untreated ILVEN patients, 11 PV patients, and 8
healthy controls, but no cases of linear psoriasis. These patients were diagnosed
based on clinicopathological features10 and not associated with systemic disorders. ILVEN patients had
no personal and family histories of psoriasis. PV lesional area was <10% of the
body surface area.
We performed immunohistochemistry on paraffin-embedded sections. Primary antibodies
included goat anti-K16 (SC-49176, 1:200; Santa Cruz Biotechnology, CA, USA), mouse
anti-involucrin (SC-21748, 1:200; Santa Cruz), filaggrin (SC-66192, 1:200; Santa
Cruz), and rabbit anti-Ki-67 (RMA-0542, prediluted; Zhongshan Jinqiao Biotechnology
Co Ltd, Beijing, China). Epidermal Ki-67+ cells were reckoned in 5
different fields at × 200 magnification and expressed in mm of the basement
membrane.11 (link) The data were
analyzed using ANOVA with Dunnett T3 test.
healthy controls, but no cases of linear psoriasis. These patients were diagnosed
based on clinicopathological features10 and not associated with systemic disorders. ILVEN patients had
no personal and family histories of psoriasis. PV lesional area was <10% of the
body surface area.
We performed immunohistochemistry on paraffin-embedded sections. Primary antibodies
included goat anti-K16 (SC-49176, 1:200; Santa Cruz Biotechnology, CA, USA), mouse
anti-involucrin (SC-21748, 1:200; Santa Cruz), filaggrin (SC-66192, 1:200; Santa
Cruz), and rabbit anti-Ki-67 (RMA-0542, prediluted; Zhongshan Jinqiao Biotechnology
Co Ltd, Beijing, China). Epidermal Ki-67+ cells were reckoned in 5
different fields at × 200 magnification and expressed in mm of the basement
membrane.11 (link) The data were
analyzed using ANOVA with Dunnett T3 test.
9
Immunostaining of Skin Cryosections
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Skin samples were fixed in 4% paraformaldehyde,dehydrated in 30% sucrose, embedded in OCT and frozen. Skin cryostat sections (5mm) were stained with antibodies specific for YFP (life technologies, Grand Island, NY), OX40 (BD Biosciences), filaggrin (Santacruz, Dallas, TX), involucrin (Covance, Princeton, NJ), loricrin, cytokeratin14, cytokeratin15, cytokeratin10 (Genetex, Irvine, CA) Ki67, CD4 (eBioscience), cleaved caspase 3 (Cellsignaling), and TSLP (R&D Systems, Minneapolis, MN), then stained with fluorescently-coupled secondary anti-rabbit or -rat IgG (Cellsignaling Technologies, Danvers, MA), or streptavidin-conjugated Cy5 (Biolegend). Images were obtained using LAS AF 6.2 software on a motorized Leica DMI 6000B fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
10
Immunohistochemical Analysis of Mouse Skin
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Mouse skin tissues were fixed with 4% PFA and embedded in paraffin, and 6 μm sections were prepared and stained with Lin28a (1:500, Abcam, UK), keratin 1 (CK1) (1:1000, Santa Cruz Biotechnology, TX, USA), keratin 10 (CK10) (1:10,000, Abcam), filaggrin (1:1000, Santa Cruz Biotechnology), involucrin (1:1000, Santa Cruz Biotechnology), and Ki67 (1:200, Abcam), which analyzed using immunohistochemistry methods. The VECTASTAIN ABC kit (Vector Labs, CA, USA) was used for anti-rabbit and anti-mouse antibodies. Signals were detected using 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate peroxidase substrate (DAB, Vector Labs) staining.
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