Phospho stat3

Cells were lysed with RIPA Lysis and Extraction Buffer (89901, Thermo Fisher, Waltham, MA, USA) including Halt^TM Protease Inhibitor Cocktail (78410, Thermo Fisher) and protein concentrations were determined using the Bradford method (Molecular Devices, Downingtown, PA, USA). Protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Proteins were incubated with antibodies against GRIM19 (ab110240, Abcam, Cambridge, MA, USA), phospho-STAT3 (Tyr705; 9145, Cell Signaling, Danvers, MA, USA), phospho-STAT3 (Ser727; 9134, Cell Signaling), STAT3 (Cell 9139, Signaling), and β-actin (sc-47778, Santa Cruz Biotechnology). They were then detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA) and HyperFilm (Agfa, Mortsel, Belgium), with β-actin used as a loading control.

For western blotting, we used the following antibodies: phospho-STAT3 (Tyr705, No.9145); phospho-STAT3 (Ser727, No.9134; Cell Signaling Technology, Danvers, MA); and Hsp90ɑ/β (F-8, No.SC13119; Santa Cruz Biotechnology, CA). For immunofluorescence staining, we used an antibody against STAT3 (H-190, No.SC7179; Santa Cruz Biotechnology).

Recombinant human IL-26 protein (MBS1362134) was purchased from MyBiosource (San Diego, CA, USA). Palmitate (P0500), metformin (D150959), methylthiazolyldiphenyl-tetrazolium bromide (MTT) (M5655), and bovine serum albumin (BSA) (A7030) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Antibodies against cyclooxygenase-2 (COX-2; 1:1000; 35-8200) were obtained from Thermo Fisher Scientific (Waltham, MA, USA), while those against collagen type II (COL-II; 1:1000; ab34712) and matrix metalloproteinase-1 (MMP-1) (1:1000; ab134184) were purchased from Abcam (Cambridge, MA, USA), and those against IL-6 (1:1000; #12153), Erk1/2 (1:1000; #4695), phospho-Erk1/2 (1:1000; #9101), phospho-c-Jun (1:1000, #3270), phospho-p38 (1:1000, #9211), phospho-JNK (1:1000, #9251), STAT1 (1:1000, #14994), phospho-STAT1 (1:1000, #9131), STAT3 (1:1000, #9139), phospho-STAT3 (1:1000, #9131), histone H3 (1:2000, #9715), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; #5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Pre-designed small interfering RNA (siRNA) directed against human TCHHL1 (s43057 and s43059) and Kruppel-like factor 4 (KLF4; s17795) and negative control siRNA were purchased from Life Technologies (Carlsbad, CA). An epidermal growth factor receptor (EGFR) inhibitor (AG 1478) was purchased from Abcam (Cambridge, UK). An antibody against an oligopeptide (HPQRERLVLQREASTTKQ) corresponding to part of the C-terminal region of TCHHL1 was previously generated^{12 (link)}. Antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2; #4695), phospho-ERK1/2 (#4370), stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK; #9252), phospho-SAPK/JNK (#4668), p38 mitogen-activated protein kinase (p38 MAPK; #8690), phospho-p38 MAPK (#4511), v-akt murine thymoma viral oncogene homolog (AKT; #9272), phospho-AKT (#9271), signal transducers and activator of transcription 3 (STAT3; #9132), phospho-STAT3 (#9145), EGFR(#4267), phospho-EGFR (#4407) and β-Actin (#4967) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Antibodies against cytokeratin-10 (M7002), Ki67 (M7240), and p53 (M7001) were purchased from DAKO (Carpinteria, CA). Antibodies against human cytokeratin-14 (ab7800) and filaggrin (ab218863), and antibodies against transglutaminase1 (PA5-59088) were purchased from Abcam and Thermo Fisher Scientific Inc. (Waltham, MA), respectively.