Anti involucrin

Antibody staining of cells was performed in the same way as described in siRNA Screening and Transfection. Plates were washed once with PBS and fixed with 4% paraformaldehyde incubated for 10 min at room temperature. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature, incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) or anti-cleaved caspase 3 (Asp175) (Cell Signaling catalog no. 9661) diluted to 1 µg/mL or according to manufacturer’s instructions in blocking buffer. Plates were then washed three times with PBS, stained with the secondary antibodies Alexa Fluor 555 donkey anti-mouse (Thermo Fisher Scientific) and/or Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific) at 1 µg/mL, the nuclear dye DRAQ5 (abcam) at 10 µM, and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) at 12.6 nM in blocking buffer. Secondary stains were incubated for 2 h at room temperature protected from light, and plates were finally washed three times with PBS before being imaged using the Perkin-Elmer Operetta High-Content Imaging System.

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.