Anti filaggrin

A six-well plate was used to culture HaCaT cells or NHEK cells for 24 h, followed by 12 h, 24 h, 48 h, or 72 h of UPF treatment at the above-mentioned concentrations along with controls. Following cell harvest, the total protein extracted from each culture was resolved on 8% SDS-PAGE. This was followed by transfer to polyvinylidene difluoride (PVDF) membrane and overnight incubation with the following antibodies at 4 °C: with rabbit anti-phospho-p120-catenin, anti-p120-catenin, anti-phospho-PLCγ1, anti-PLCγ1, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-CaSR, anti-Involucrin or anti-tubulin antibody (Cell Signaling Technology, Beverly, MA, USA), and with anti-Filaggrin (Santa Cruz Biotechnology, Dallas, TX, USA) for overnight. Post addition of goat anti-rabbit antibody or goat anti-mouse antibody (Cell Signaling Technology, Beverly, MA, USA) respectively, the detection was done using a chemiluminescence substrate (Pierce, Rockford, IL, USA) followed by the use of Amersham Imager (GE Healthcare Biosciences, Pittsburgh, PA, USA) to record images.

Citrus junos seed residue, a byproduct of the post-yuzu-juice-preparation process, was obtained from the Lotte R&D Center (Seoul, Republic of Korea). The following monoclonal antibodies were used: anti-tyrosinase-related protein-2 (TRP-2) (Abcam, Cambridge, UK), anti-collagen type IV (Abcam), and anti-filaggrin (Santa Cruz Biotechnology, Dallas, TX, USA). Anti-collagen type I antibody and all cell culture solutions were purchased from Invitrogen (Carlsbad, CA, USA).
For Western blotting, antibodies that act against tyrosinase, TRP-1, TRP-2, and MITF were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), whereas antibodies that act against α-MSH, melanin, and β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Briefly, 96-well microtiter plates were coated with polyclonal anti-collagen (Abcam, Cambridge, UK), anti-filaggrin (Santa Cruz Biotechnology, Dallas, Texas, USA), or anti-fibronectin (Abcam) antibodies (1:100 dilution) in 100μl of coating buffer (eBioscience) and incubated overnight at 4°C. The plates were washed seven times with PBS-T and incubated with assay buffer (eBioscience) for 1 h at room temperature (RT). The plates were again washed seven times with PBS-T. Serum samples (1:10 diluted in PBS) were added to each well in triplicate, and the plates were incubated for 2 h at RT. The plates were washed seven times with PBS-T, and then the 12G1 mAb (1:100 diluted in assay buffer) was added, and the plates were incubated for 2 h at RT. After incubation, the plates were washed seven times in PBS-T, 50µl of HRP-conjugated anti-mouse IgG antibody (1:3000, Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) was added, and the plates were incubated for a further 1 hour at 37 °C. Finally, the plates were washed nine times, and the bound antibodies were visualized by adding substrate solution (eBioscience). Reactions were stopped after 15 mins by adding 50µl of stop solution (eBioscience). The colorimetric reaction was measured at 450 nm on a VersaMax ELISA reader.

The BCA protein assay kit (Aspen Biotechnology, Wuhan, China) was used to determine protein concentration. Equal protein amounts (30 μg/lane) were separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk for 1 h at room temperature, and incubated at 4° C overnight with the following primary antibodies diluted 1:1000: anti-p27 (ab32034), anti-CDK4 (ab108357), anti-keratin 1 (ab185628), anti-keratin 5 (ab64081), anti-keratin 10 (ab237775), Anti-filaggrin (sc-66192), anti-loricrin (ab137533), anti-p-STAT3 (phospho Y705) (ab76315), anti-cyclin D2 (ab207604), and anti-p-JAK1 (ab125051). Subsequently, the membranes were incubated with the corresponded secondary antibodies (1:5000) at room temperature for 1 h. Finally, the ECL kit (Thermo Fisher Scientific) was used to visualize the protein bands. Anti-filaggrin was provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Other antibodies were purchased from Abcam (Cambridge, MA, USA).