Ab194898

Mammary glands from female Cdkn1b^+/+ and Cdkn1b^-/- groups (both N1xN1 and N6xN6) were harvested and whole mounts were generated as described previously [30 (link)]. The inguinal and abdominal mammary glands were collected, fixed, stained in carmine, dehydrated and cleared in xylene. Samples were imaged on a Nikon Ti/E inverted microscope using Nikon Elements software. Histology and multicolor immunofluorescence analyses were performed as described previously [3 (link)]. After heat-induced antigen retrieval in TRIS-EDTA buffer (pH 9), the samples were blocked with 5% goat serum PBS and stained with antibodies against Ki67 (BD550609, 1:50), Milk-specific proteins (Nordic-MUbio, RAM/MSP, 1:200), PR (ab16661, 1:100), FoxA1 (ab23738, 1:200) and phospho-Stat5 (AbCam ab194898, 1:250). Images from the stained sections were obtained by Nikon inverted widefield live-cell imaging system. The percentage of cells for each marker was estimated by counting positive cells and total cells per field from 4 to 6 randomly selected regions using ImageJ 1.45s software. Luminal ectasia was scored by measuring the percentage area of milk protein to mammary epithelium.

TSN was purchased from Pusi Bio-Technology. MG132 (MB5137) and lenalidomide (01131085) were obtained from Meilunbio and Adamas-Beta, respectively. Phytohemagglutinin (PHA, abs47014909), phorbol myristate acetate (PMA, abs9107), and ELISA kits for IL-2 (abs551102) were all obtained from Absin Bioscience Inc. Antibodies for STAT1 (ab109461, 1:1,000), STAT2 (ab32367, 1:1,000), STAT4 (ab68156, 1:1,000), STAT5 (ab194898, 1:1,000), STAT6 (ab32520, 1:1,000), Bcl2 (ab182858, 1:1,000), c-Myc (ab32072, 1:1,000), and ubiquitin (ab134953, 1:500) were obtained from Abcam. Antibodies for STAT3 (9139s, 1:1,000), p-STAT3 (Y705) (9145T, 1:1,000), PD-L1(13684, 1:1,000), cyclin D1 (55506, 1:1,000), BCL-XL (2764, 1:1,000), Bim (2933, 1:1,000), HRP-linked anti–rabbit IgG (7074, 1:5,000), HRP-linked anti–mouse IgG (7076, 1:5,000), and Alexa Fluor 647 conjugate (4418, 1:1,000) were from Cell Signaling Technology. WT mouse IgG (sc-2025, 1:50) was obtained from Santa Cruz Biotechnology Inc. Annexin V-FITC apoptosis assay kit was purchased from Absin, cell cycle and apoptosis analysis kit from was purchased from Beyotime, and Neofect DNA transfection reagent (TF20121201) was provided by Beijing SBS Genetech Co. Ltd. Recombinant human STAT3 protein (P40763) was obtained from Novoprotein.