Ab32512

The uterine tissue was homogenized in radioimmunoprecipitation assay buffer. And then the homogenate was centrifuged at 13,000 rpm for 20 min at 4°C and the supernatant was collected. The protein concentration was determined using the bicinchoninic acid method (BCA) according to the kit's instruction (02912E, Kangwei Century, Beijing, China), and 20 mg of protein was loaded in each lane. Protein samples were separated on 5 to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and electrophoretically transferred to polyvinyl difluoride (PVDF) membranes (Millipore, MO, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 hour, followed by overnight incubation at 4°C with the following primary antibodies diluted in blocking buffer : anti-CCR3 (1 : 1000, ab32512, Abcam) and anti-HR1R (1 : 500, BS-6663R, Bioss, Beijing, China). Subsequently, the immunoblots were incubated with second antibodies (1 : 10000, 111-035-003, Jackson, PA, USA) for 1 hour at room temperature. GAPDH (1 : 1000, 5174, CST, MA, USA) was used as an internal control. The immunoreactivity was detected by an ECL chemiluminescence detection system (WBKLS0500, Millipore) and analyzed by an image analysis system (Tanon 4200, Shanghai, China).

Cell samples were fixed with 4% PFA for 15 min at RT. Tissues were fixed with 4% PFA overnight at RT. Tissue samples were embedded in paraffin and sliced into 5 mm sections. Samples were incubated with following antibodies: Piezo1 (ab128245, 1:100, Abcam), α-SMA (ab7817, 1:100, Abcam), CCL24 (22306-1-AP, 1:200, Proteintech), CCR3 (ab32512, 1:100, Abcam), Wnt2 (A5864, 1:200, Abclonal, Wuhan, China), Wnt11 (ab31962, 1:200, Abcam), β-catenin(#8480, 1:100, CST), Alexa Fluor 594 goat anti-rabbit secondary antibody (Jackson lab, 125369, 1:200) and Alexa Fluor 488 goat anti-mouse secondary antibody (Jackson lab, 133384, 1:200). Images were visualized using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).

Patients were grouped by their response to ADT into three groups: (1) Favourable Sustained Response to ADT (SRADT) with PSA remaining at nadir levels and below 4 ng/ml throughout the follow up period, (2) Initial Response to ADT (IRADT) with PSA falling below 4 ng/ml but then developing CRPC, and (3) Poor Response to ADT (PRADT) with nPSA not falling below 4 ng/ml and subsequently developing CRPC. Formalin fixed paraffin embedded prostate tumour sections were obtained in accordance with appropriate ethics approval (National Health Service Research Ethics Committee permission: 12/EE/0058), including SRADT (n = 6), IRADT (n = 6), and PRADT (n = 6). Sections were stained for CCR3 as a secondary outcome measure of PPFV influence on prostatic epithelium (rabbit monoclonal antibody; Abcam, ab32512 1:50 dilution), and signal amplified with Dako Rabbit EnVision (K4003) and visualised using Dako Liquid 3,3′-diaminobenzidine (DAB; K3468). Using the Leica Biosystems Image Analysis suit (v4.0.6), DAB staining was quantified by normalising the area of DAB staining to the area of haematoxylin counterstain.

The western blot analysis was performed as described in our previous study^{20 (link)}. The primary antibodies used in the this study were listed as follows: rabbit anti-gankyrin (ab182576), rabbit anti-STAT3 (ab32500), and rabbit anti-STAT3 (Phospho 727) (ab30647), rabbit anti-eotaxin-2 (CCL24) (ab203586), rabbit anti-CCR3 (ab32512) from Abcam (Cambridge, MA, USA) and rabbit anti-GAPDH (#2118S), rabbit anti-p44/42 MAPK (Erk1/2) (#4695), rabbit anti-p44/42 MAPK (Erk1/2) (Phospho Thr202/Tyr204) (#4370), rabbit anti-Akt (#9272), and rabbit anti-Akt (Phospho Ser473) (#9271) from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody used in the assay was anti-rabbit IgG-HRP-linked antibody (#7074S) from Cell Signaling Technology. Coimmunoprecipitation analysis was performed according to previously published protocols^{20 (link)} using the above-mentioned antibodies.