Py705 stat3

Antibodies to p-ERK1/2, ERK1/2, p-T308-AKT, AKT, p-Y705-STAT3, JAK1 were purchased from Cell Signaling Technology (Beverly, MA, USA). STAT3 and p-T1022-JAK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to HAS2 and C5R1 were purchased from Abcam (Cambridge, UK). 4,6-diamidino-2-phenylindole (DAPI) were purchased form Sigma (St Louis, MO, USA). rh-C5a protein and antibodies to CD105 and C5a were purchased from R&D Systems (Minneapolis, MN, USA). Chemical inhibitors specific to MEK (U0126), PI3K (LY294002), JAK1 (P6) and STAT3 (WP1066) were purchased from Calbiochem (San Diego, CA, USA). Anti-Goat Alexa Fluor 488, anti-mouse Alexa Fluor 546 were purchased from Invitrogen (Carlsbad, CA, USA). Anti-mouse IgG-HRP, anti-goat IgG-HRP and anti-rabbit Ig-HRP were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). HA (Low molecular weight; 15-40 kDa) was purchased from R&D systems (Minneapolis, MN, USA).

Liver tissues from human liver cancer patients, Tg^TM4SF5 FVB/N mice, or C57BL/6 (WT or Tm4sf5^−/− KO) mice treated with or without DEN and in the absence or presence of TSAHC were processed for immunohistochemistry. Liver sections were fixed with 3.7% formaldehyde and embedded in paraffin. The fixed liver sections were deparaffinized and rehydrated. Antigen retrieval was performed with heat-induced epitope retrieval (HIER) using sodium citrate buffer (pH 6.0). Quenching and blocking were performed using 3% H₂O₂ in distilled water and 1% normal goat serum in phosphate-buffered saline (PBS). Antigens were stained using the avidin–biotin complex (ABC) method (VECTASTAIN Elite ABC HRP Kit, Vector) and were detected using 3,3′-diaminobenzidine (DAB) stain (Vector). Antibodies against TM4SF5 [17 (link)], collagen I (Acris Antibodies), pY⁷⁰⁵STAT3 (Cell Signaling Technology), normal rabbit or mouse IgG, α-SMA (Sigma-Aldrich), α-fetoprotein (AFP), CD34, Ki67, laminins (Abcam), α-l-fucosidase [FUCA (AFU)], and laminin γ2 (Santa Cruz Biotechnology) were used for immunostaining. Ten random images per slide were saved using a digital slide scanner (MoticEasyScan, Motic, British Columbia, Canada). The tissues were also processed with Masson’s trichrome as well as hematoxylin and eosin stains, as previously described [27 (link)].

Cell lysates were prepared in PN lysis buffer (10 mM PIPES, 50 mM NaCl, 150 mM sucrose, 50 mM NaF, 40 mM Na₄P₂O₇10H₂O, 1 mM Na₃VO₄, 1% Triton X-100, Complete protease inhibitors (Sigma)). Total protein (30 µg) was separated by SDS-PAGE, and transferred to nitrocellulose using the Trans-Blot Turbo Transfer System (Bio-Rad). Blots were blocked in blocking buffer 5% bovine serum albumin (BSA, Fisher) in TBS-T (TBS-T is 50 mM Tris-Cl, 150 mM NaCl, pH 8 with 0.1% Tween20) for 1 hour at 22 ^oC, then incubated overnight at 4 ^oC with primary antibodies diluted in blocking buffer. Blots were further incubated with secondary goat anti-mouse or -rabbit fluorophore-conjugated antibodies (Dylight 800 or Dylight 680, ThermoFisher) in 2% non-fat milk in TBS-T, and scanned on the Licor Odyssey.
The following primary antibodies were used: pY705-STAT3, pS727-STAT3, and STAT3 (Cell Signaling Technologies); and GAPDH (Biolegend). In some experiments, cells were treated with IL-6 at the indicated concentrations and time prior to preparation of cell lysates.