Anti keratin 8

After the indicated stimulation, cells were fixed with cold methyl alcohol for 5 min at −20 °C. Cells were then permeabilized in phosphate buffer solution (PBS, pH7.4) containing 5% goat serum and 0.1% Triton X-100 for 60 min at room temperature. Next, the monoclonal antibodies anti-keratin-18, anti-keratin-8, anti-keratin-8-Ser431, anti-keratin-8-Ser73, anti-keratin-18-Ser52 (Abcam, Cambridge, UK), or anti-pan-keratin (Cell Signaling Technology, USA) were added overnight at 4 °C. Cells were then incubated with FITC (fluorescein isothiocyanate isomer)-conjugated or Alexa Flour 594-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h without light. After washing with PBS, imaging was performed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 100× objective.

The following antibodies were used: anti-FLAG (F1804; Merck), anti-keratin 5 (RM-2106-S0; Thermo Fisher Scientific), anti-keratin 8 (ab53280; Abcam, Cambridge, UK), anti-keratin 14 (MS-115-P0; Thermo Fisher Scientific), anti-keratin 18 (MS-142-P0; Thermo Fisher Scientific), anti-SON (HPA023535; Merck), anti-FAM83H (HPA024604; Merck), anti-CK1α (sc-6477; Santa Cruz Biotechnology, CA, USA), anti-GAPDH (2275-PC-100; R&D Systems, MN, USA), and anti-SC-35 (S4045; Merck). Alexa Fluor 488-conjugated donkey anti-mouse IgG (A-21202) and donkey anti-rabbit IgG (A-21206), Alexa Fluor 568-conjugated donkey anti-mouse IgG (A10037) and donkey anti-rabbit IgG (A10042), Alexa Fluor 594-conjugated donkey anti-goat IgG (A-11058), and Alexa Fluor 647-conjugated donkey anti-mouse IgG (A-31571) were purchased from Thermo Fisher Scientific. Horseradish peroxidase-conjugated anti-mouse IgG (#7076; Cell Signaling Technology, MA, USA) and anti-rabbit IgG (711-035-152; Jackson Immunoresearch, PA, USA) antibodies were used for Western blotting.