Following treatment with stimulatorα, HaCaT cells were harvested and lysed in lysis buffer. Samples were separated by performing 10% SDS-PAGE and then transferred to nitrocellulose membrane. Blots were incubated with antibodies against filaggrin, phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), involucrin, or loricrin (Proteintech, Rosemont, IL, USA). After incubation, the membrane was developed by using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Loricrin
Manufactured by Proteintech
Sourced in United States
Loricrin is a lab equipment product from Proteintech. Loricrin is a structural protein that plays a crucial role in the formation of the cornified cell envelope in the upper layers of the epidermis.
Automatically generated - may contain errors
Lab products found in correlation
Involucrin, by Proteintech (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence detection system, by Cytiva (1 mentions)
Phospho jnk, by Santa Cruz Biotechnology (1 mentions)
Filaggrin, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
3 protocols using loricrin
1
Immunoblot Analysis of Epidermal Differentiation Markers
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
Protein Expression Analysis of Epidermal Markers
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Cells in the LV, NC, and Blank groups were collected, washed twice in ice-cold PBS,
and then lyzed and homogenized using lysis buffer for 30 min on ice. The resulting
samples were diluted 1:1 with 2× sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) loading buffer, followed by thermal denaturation at 100°C
for 5 min. After cooling, the supernatants were centrifuged at 10,000
g for 10 min at 4°C to remove the insoluble precipitate. The
samples were separated on 10% SDS-polyacrylamide gels, followed by 2 h blocking with
blocking buffer. After that, the membranes were incubated overnight at 4°C with
primary antibodies, and then with secondary antibodies for 2 h at room temperature,
followed by enhancement with a chemiluminescence reagent (Pierce, USA) in the dark.
The level of protein expression was quantified by the Gel-Pro Analyzer software
(Media Cybernetics, USA). GAPDH was used as the internal reference. The following
polyclonal primary antibodies were obtained from GeneTex (Irvine, USA): rabbit
anti-human cytokeratin 5 (CK-5, Cat #GTX113219), cytokeratin 10 (CK-10, Cat
#GTX108883), cytokeratin 14 (CK-14, Cat #GTX104124), loricrin (Cat #GTX116013), and
involucrin (Cat #GTX116012); rabbit anti-human transglutaminase-1 (TGM-1) polyclonal
antibody (Cat #12912-3-AP) was from Proteintech (USA).
and then lyzed and homogenized using lysis buffer for 30 min on ice. The resulting
samples were diluted 1:1 with 2× sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) loading buffer, followed by thermal denaturation at 100°C
for 5 min. After cooling, the supernatants were centrifuged at 10,000
g for 10 min at 4°C to remove the insoluble precipitate. The
samples were separated on 10% SDS-polyacrylamide gels, followed by 2 h blocking with
blocking buffer. After that, the membranes were incubated overnight at 4°C with
primary antibodies, and then with secondary antibodies for 2 h at room temperature,
followed by enhancement with a chemiluminescence reagent (Pierce, USA) in the dark.
The level of protein expression was quantified by the Gel-Pro Analyzer software
(Media Cybernetics, USA). GAPDH was used as the internal reference. The following
polyclonal primary antibodies were obtained from GeneTex (Irvine, USA): rabbit
anti-human cytokeratin 5 (CK-5, Cat #GTX113219), cytokeratin 10 (CK-10, Cat
#GTX108883), cytokeratin 14 (CK-14, Cat #GTX104124), loricrin (Cat #GTX116013), and
involucrin (Cat #GTX116012); rabbit anti-human transglutaminase-1 (TGM-1) polyclonal
antibody (Cat #12912-3-AP) was from Proteintech (USA).
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