Anti stat3 c 20 sc 482

Western blot and immunostaining were performed using standard protocols [26 (link)]. Anti-LOX-1 (LOX19-22) sc-66155, anti-AR (N-20) sc-816, anti-GAPDH (0411) sc-47724, anti-NF-kB p65 C-20) sc-372 and anti-IκB-alfa (C-21) sc-371, and anti-STAT3 (c-20) sc-482 antibodies were obtained from Santa Cruz Biotechnology. Anti-p-p65 s536 7F1, anti-p-IκB-alfa S32/S36 5A5, androgen receptor (AR-V7 Specific), and anti-p-STAT3 (Y705) (D3A7) antibodies were obtained from Cell Signaling. For Western blot, the anti-mouse IgG-Alexa Fluor 680 and anti-rabbit IgG-Alexa Fluor 790 secondary antibodies were obtained from Jackson ImmunoResearch. Western blots were analyzed using LICOR-CLX. An anti-rabbit IgG/FITC secondary antibody from Jackson ImmunoResearch was used for immunofluorescences. Photographs were obtained by confocal microscopy (Olympus IX81, Japan). To determine IL-6 concentration from supernatants, the human ELISA Kit (D6050, R&D) was used according to manufacturer’s instructions.

Protein extract preparation and immunoblotting were performed accordingly to standard procedures [36 (link),38 (link)]. The Abs used for the study were as follows: anti-PI3Kα (#4249S), anti-PI3Kβ (#3011S), anti-PI3Kδ (#34050S), anti-p-STAT3 (Tyr705#9131S), anti-p-AKT (T308#9275S and S473#9271S), anti-p-PDK1 (S241#3061S), anti-PDK1 (#3062S), anti-p-S6 (S235/236#2211), anti-S6 (#2317S), and anti-p-p65 (S276#3033S) (all from cell signaling); anti-K10 (#PRB-159P) and anti-Loricrin (PRB-145P) (both from Covance); anti-cyclin D1 (#sc-20044), anti-STAT3 (C-20#sc-482), and anti-β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-keratin 5 (K5) (#MA5-14473, Invitrogen). Filters were properly developed with anti-mouse, anti-goat, or anti-rabbit Ig Abs conjugated to HRP using the ECL-plus detection system (Amersham, Dubendorf, Switzerland), or, otherwise, the SuperSignal West Femto kit (Pierce, Rockford, IL, USA). Immunoblots were subjected to densitometry using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA) supported by the Molecular Analyst software (https://imagej.nih.gov/ij/, accessed on 20 July 2021). Band intensities were evaluated in three independent experiments and reported as means of densitometric intensity (D.I.) ± SD.