Ab85125

Western Blot analysis was performed as previously described [48 (link)] using the following primary antibodies: PIWIL1 (ab85125; Abcam, Cambridge, UK), PIWIL2 (ab98852; Abcam) and PIWIL4 (ab111714; Abcam) and α-tubulin (A9044; Sigma).

Five-μm-thick transverse sections of FFPE tissues were serially cut and mounted onto Dako Silanized Slides (Dako, Glostrup, Denmark). The immunohistochemical assay was performed as previously described [49 (link)] using rabbit polyclonal antibodies anti-human PIWIL1 (ab85125; Abcam), PIWIL2 (ab98852; Abcam) and PIWIL4 (NBP1-83491; Novus Biologicals, Littleton, CO, USA).

MCF-7 (ATCC HTB-22), SKBR3 (ATCC HTB-30) and ZR-75.1 (ATCC CRL-1500) cell lines were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (HyClone, Cramlington, UK) and antibiotics: 100U/ml penicillin, 100mg/ml streptomycin, 250ng/ml Amfotericin-B (exponential growing condition). MCF10A (ATCC CRL-10317) mammary epithelial cells were maintained in MEGM Bullet Kit (Lonza, Milan, Italy) supplemented with 100 ng/ml cholera toxin (Sigma-Aldrich, Milan, Italy). Steroid deprivation (starvation) was performed by culturing cells in phenol red-free DMEM and 5% Dextran-Coated Charcoal stripped serum (DCC-FBS) for 5 days, as described earlier [24 (link)]. PIWIL proteins expression was analyzed by sodium dodecyl sulphate (SDS) acrylamide gel electrophoresis and immunoblotting of total protein extracts, using rabbit anti-PIWIL1 (ab85125, Abcam, Cambrige, UK), rabbit anti-PIWIL2 (ab26408, Abcam), mouse anti-PIWIL3 (ab77088, Abcam), rabbit anti-PIWIL4 (ab111714, Abcam) and mouse anti-β-actin (A1978, Sigma Aldrich).