Mouse anti ar

Laemmli sample buffer (Sigma-Aldrich) was used to collect protein samples. Lysates were heated to 100 °C for 10 min. Samples were loaded onto precast SDS-PAGE gels (Bio-Rad) and run until completion. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF) using a semi-dry blotter (Bio-Rad). Membranes were blocked at room temperature for 1 h using LI-COR “Intercept” Blocking Buffer. Blots were incubated with primary antibody at 4 °C overnight in LI-COR “Intercept” Antibody Buffer for immunodetection. Primary antibodies are mouse anti-AR (1:1000; #sc-166918; Santa Cruz Biotechnology Inc., Dallas, TX), rabbit anti-Iba1 antibody (1:1000; #019-19741; Wako), rabbit anti-RBPMS primary antibody (1:500; #1830; PhosphoSolution), rabbit p-p38 (1:500; #4511S; Cell signaling Technology, Inc.), p38 (1:1000; #8690S; Cell signaling Technology, Inc.), GFAP (1:1000; #6370S; Cell signaling Technology, Inc.),)and GAPDH (1:1000; #2118S; Cell signaling Technology, Inc). The membrane was incubated with species-specific secondary antibodies linked to near IR dyes (LI-COR, IRDye 680RD donkey anti-mouse (#926-68072), IRDye 800CW Goat anti-rabbit (#926-3211) at 1:10,000 dilution for 4 h at room temperature, washed, and imaged on a LI-COR Odyssey IR in a linear range.

The extraction of cellular proteins and their detection via western blotting were performed as previously described [21 (link), 24 (link)]. Western blotting was performed using the following primary antibodies: mouse anti-AR, mouse anti-beclin1, goat anti-LC3β, mouse anti-LAMP1, and rabbit anti-IKKγ antibodies (1:500, Santa Cruz Biotechnology); mouse anti-TLR4, rabbit anti-iNOS, rabbit anti-IKKα, and rabbit anti-4-HNE (1:1000, Abcam); rabbit anti-IKKβ and rabbit anti-phospho-IκBα antibodies (1:1000, Epitomics); rabbit anti-phospho-IKKα/β, rabbit anti-IκBα, rabbit anti-p65, and rabbit anti-phospho-p65 antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA); and mouse anti-β-actin antibodies (1:8000, Sigma-Aldrich). The blots were then incubated with their respective secondary antibodies: horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (both 1:8000, Abcam), and donkey anti-goat IgG (1:5000, Santa Cruz Biotechnology). β-actin was used as the loading control. The immunoreactive bands were scanned using the Bio-Rad ChemiDoc™ XRS⁺ imager with Image Lab™ Software (Bio-Rad Laboratories, CA, USA). Band intensity was quantified using Quantity One software (Bio-Rad Laboratories).

LNCaP cells were starved overnight, then transfected with DsiRNAs for 48 h. Cells were fixed in ice-cold methanol for 4 min at –20 °C after 4 h of stimulation with DHT (10 nM). This was followed by three washes with PBS and incubation with the following antibodies diluted in blocking buffer (0.2% BSA (Bioshop), 0.1% Triton X100 (Sigma-Aldrich)): rabbit anti-FLAG (Sigma Aldrich, #F7425), mouse anti-AR (Santa Cruz, #7305), or rabbit anti-KLF4 (Abcam, #215036) for 1 h at room temperature. After washes in PBS, coverslips were incubated with Alexa 568-conjugated goat anti-rabbit (Thermo Fisher Scientific, #A11011) or Alexa 488-conjugated goat anti-mouse (Cell Signalling Technology, #4408) antibodies for 1 h at room temperature. They were washed twice with PBS before being mounted on slides using ProLong Gold antifade with DAPI (Thermo Fisher). Pictures were acquired with an Olympus FV1000 using the FluoView 3.0 software or with a Nikon Eclipse E600 imaging system using MetaView.