Anti pstat3 t705

Formalin fixed and paraffin embedded RM-1 mouse tumors were subjected to immunohistochemistry (IHC) using Dako Autostainer Plus En VisionTM + Kit (Dako, Glostrup, Denmark) and stained with the antibodies anti-pSTAT3-T705 (ab76315 Abcam, Cambridge, UK), anti-pSTAT3-S727 (#9143 Cell Signaling Technology, Danvers, MA, USA), anti-FOXP3 (ab54501 Abcam) and anti-CD45 (ab25386 Abcam). The immunostainings were analyzed using Halo image analysis software (Indica Laboratories, Albuquerque, NM, USA).
For immunofluorescent staining, the RM-1 tumors were stained using the Opal Multiplex immunofluorescence assay (Akoya Biosciences) and imaged with Mantra multispectral image system (Akoya Biosciences). Antibodies used were anti-CD3E (LS-C343957 LSBio), anti-CTLA-4 (ab 237,712 Abcam) and anti-pSTAT3-S727 (#9143 Cell Signaling Technology).

LNCaP and C4–2 cells were treated with GPB730 and enzalutamide for 72 h. Cell lysates were prepared and western blot analysis was performed according to previous publication [21 (link),22 (link)]. Primary antibodies used were anti-STAT3 (#4904 Cell Signaling Technology), anti-pSTAT3-T705 (ab76315 Abcam), anti-pSTAT3-S727 (#9134 Cell Signaling Technology), c-myc (ab32072 Abcam), survivin (#2808 Cell Signaling Technology), PSA (sc7638 Santa Cruz) and androgen receptor (ab108341 Abcam). Beta-actin (#A5441 Sigma) was used as loading control. LNCaP cells stimulated with 50 ng/ml IL-6 for 30 min and DU145 cells were used as positive controls for pSTAT3-S727 and pSTAT3-T705 expression.