To harvest tissues for immonoblotting, tissues were lysed in Pro-Prep solution (Intron, Daejeon, Korea) and homogenized with the TissueLyser (Qiagen, Hilden, Germany). To harvest cell lysates for immunoblotting, cells were lysed in Pro-Prep solution. Total protein was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Samples (20~30 µg protein per lane) were run on sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with the appropriate antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Intron). The following primary antibodies were used for western blots: S100A8, involucrin, and actin (Santa Cruz Biotechnologies), loricrin (Covance, Princeton, NJ, USA), and filaggrin (Abcam, Cambridge, UK).
Involucrin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Involucrin is a structural protein found in the upper layers of the epidermis. It is a key component of the cornified envelope, which provides mechanical and barrier functions to the skin. Involucrin contributes to the formation of the protective barrier that helps maintain skin integrity.
Automatically generated - may contain errors
Lab products found in correlation
Filaggrin, by Santa Cruz Biotechnology (3 mentions)
Filaggrin, by Abcam (2 mentions)
β catenin, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Cytokeratin 10, by Santa Cruz Biotechnology (2 mentions)
Ly 6g ly 6c, by BioLegend (2 mentions)
Cd11b, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by BD (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Loricrin, by Fortrea (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Ube2c, by Abcam (1 mentions)
Akt inhibitor ly294002, by Cell Signaling Technology (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
17 protocols using involucrin
1
Immunoblotting Analysis of Skin Proteins
2
Immunoblotting Analysis of Stemness and EMT Markers
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Cell lysates were prepared after 48 hrs of transfection in NP-40 lysis buffer (Invitrogen) and protease inhibitor cocktail (1X). Equivalent amounts of denatured protein samples were subjected to SDS-PAGE (8%-10%), separated by size and transferred on to PVDF membrane (Millipore, Billerica, USA). Antibodies used for immuno-blotting were polyclonal β-catenin, E-cadherin, CD44 and CD24, Involucrin (Santa Cruz Biotechnology, CA, USA), polyclonal Oct4 and Sox2 (Abcam), polyclonal C-myc, Akt and phospho-Akt (Cell Signaling Technology, USA) and UBE2C (Abcam). Bands were obtained using ECL substrate (Thermo Scientific, USA) from HRP-conjugated secondary antibody (Sigma). Proteasome Inhibitor MG132 (Calbiochem) and Akt inhibitor LY294002 (Cell signaling Technology, USA) were both used at a concentration of 50 µM. Transfected cells were treated for 4 hours before harvesting. Band intensities of each protein were analyzed by ImageJ to obtain densitometric values for their quantification. These were normalized to β-actin for individual experimental sets and fold change calculated. All the histograms were expressed as means ± S.D. of three different experiments and p values computed in GraphPad Prism 5 (Student’s two tailed t test).
3
Involucrin, Syndican-3, and E-Cadherin Expression Analysis
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ATRA was purchased from Sigma–Aldrich (St. Louis, MO), and was dissolved in dimethylsulphoxide (DMSO) as stock solution. Primary antibodies for Involucrin, Sydencan-3, and E-Cadherin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CD44 antibody was from ABGENT (San Diego, CA). Anti-β-actin antibody was from Cell Signaling Technology (Beverly, MA). siRNA oligos for CD44 and control siRNA-A were also from Santa Cruz Biotech. Inc. Enzymes I-SceI was from New England Biolabs (Ipswich, MA).
4
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).
5
Antibody Panel for Western Blot and IHC
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Antibodies used for Western blot analysis and immunohistochemistry were: Actin (A5441; Sigma-Aldrich), Flag (F7425; Sigma-Aldrich); Desmocollin-1 (sc18115; Santa Cruz Biotechnology, Inc.); Desmoglein-1 (sc20114; Santa Cruz Biotechnology, Inc.); Flg (PRB-417P; Covance); KLK5 (ab7283; Abcam); Involucrin (sc15230; Santa Cruz Biotechnology, Inc.); Loricrin (PRB-145P; Covance); TSLP (AF555; R&D Systems); and CD3 (ab5690; Abcam).
6
Histological and Immunofluorescent Analysis of Wound Healing
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All animals were sacrificed and the dorsal skin promptly excised. The excised patches (2 cm × 2 cm) were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 μm, and stained [hematoxylin and eosin (H&E) and Sirius Red] for microscopic examination. Immunofluorescent stains were also performed to assess expression levels of K10, K14, Ki-67, and involucrin during wound healing. The following antibodies were purchased from commercial sources: anti-K10 (Abcam, Cambridge, UK), anti-K14 (Abcam), anti-Ki-67 (Acris Antibodies, Herford, Germany), and involucrin (Santa Cruz Biotechnology, Dallas, TX, USA). Quantitative assessment of immunoreactivity was enabled by proprietary image analysis software (i-Solution; IMT Inc, https://www.IMT-Solution.com ).
7
Immunocytochemistry of Renilla and TASL KO cells
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Renilla KO and TASL KO cells were seeded into 12-well plates. ICC was performed using antibodies against CK10 (Abcam, ab76318) and Involucrin (Santa Cruz, sc-21748). The cells were then incubated with anti-rabbit IgG conjugated with Alexa Fluor 555 (Thermo Scientific, A-21428) and anti-mouse IgG Alexa Fluor 488 (Thermo Scientific, A28175). Alexa Fluor 568 Phalloidin (Thermo Scientific, A12380) was used to visualize actin polymerization in control and TASL KO cells grown in low-calcium conditions. The cells were observed using a fluorescence microscope (Leica DMI3000B, Leica, Germany).
8
Investigating Skin Barrier Regulation
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Foetal bovine serum (FBS), penicillin and Dulbecco’s modified Eagle medium (DMEM) were purchased from Hyclone (South Logan, UT, USA). Antibodies targeting β-catenin, phospho-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), phospho-Akt (Tyr525/526), Akt, phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425), Smad2/3, and β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Antibodies targeting filaggrin, loricrin, and involucrin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LPS (Escherichia coli, serotype 0111:B4) and all other chemicals were purchased from Sigma Chemical (St. Louis, MO, USA). The plasmid (β-catenin) was transfected using Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. Centella asiatica extract (CAE), as a positive control, was obtained from Korea Chong Kun Dang Pharma. Co. Ltd., in Korea.
9
Quantitative Protein Analysis Protocol
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Whole cell lysates were collected using RIPA buffer with added protease inhibitor cocktail (Roche). Insoluble fractions were collected using Urea-containing buffer. Protein was quantified using Bradford assay reagent (Biorad) and bovine serum albumin standards (Pierce). Equal amounts of protein were electrophoresed in SDS-PAGE gels and transferred to PVDF membrane Immobilon-FL (Millipore). Blots were developed using ECL-plus (GE) for 5 minutes, chemiluminescence visualized on a Licor Odyssey FL imager and quantitation done using Licor Image Studio software. All quantitated protein signals were normalized to GAPDH signal from the same gel. Antibodies used were as follows: SIRT1 (Cell Signaling), SIRT2 (Abcam), SIRT3 (Sigma), Involucrin (Santa Cruz), Cytokeratin 10(Santa Cruz), GAPDH (Santa Cruz), p-NBS1 Ser 343 (Cell Signaling), NBS1 (Santa Cruz), ac-p53 (Cell Signaling), p53 (Calbiochem), pATM Ser 1981 (Cell Signaling), ATM(Cell Signaling), pCHK2 Ser 19 (Cell Signaling), CHK2(Cell Signaling).
10
Immunohistochemical Analysis of Skin Samples
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The N-WASPHetG12D and N-WASPKOG12D mice were anesthetized and sacrificed via CO2 euthanization. Skin samples from the dorsal regions, ears and tails of the mice were processed as previously described [9 (link)]. The images were acquired using an Olympus microscope(Olympus Corporation of the Americas, Coopersburg, PA, USA) with a CoolSNAPHQ2 camera (TELEDYNE PHOTOMETRICS 3440 E. Britannia Drive, Suite 100, Tucson, AZ 85706, USA). The dilutions of the antibodies used were carried out as follows: Involucrin, K5, K10, PCNA, CD11b, SMA, CD3, and CD4 (1:100, Santa Cruz, Dallas, TX, USA.), K17, K8, K19, and K17 (1:50, DSHB Iowa, USA), Ly6G/Ly6C (1:100, Biolegend, San Diego, CA, USA), E-cadherin (1:100, BD biosciences, Singapore), K14 (1:10, LL001, a from gift from Birgit’s lab), and Alexa Fluor 594 Goat Anti-rabbit (1:50, Molecular Probes Life Tec Mass, Waltham, MA, USA).
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