Cleaved notch1

Tissue samples were homogenized in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA and 1% Triton X-100 with a complete protease inhibitor cocktail (Roche)] and then denatured in SDS sample buffer in boiling water. The total extracted proteins were separated on a 10% SDS polyacrylamide gel (Bio-Rad) and electro-transferred to a Hybond N+ membrane (GE Healthcare). Membranes were blocked with 5% (w/v) non-fat dry milk, incubated with primary antibodies against lysozyme (1:1000, Dako, A0099), NF-κB-p65 (1:1000, Cell Signaling Technology, 8242), IκBα (1:1000, 9242, Cell Signaling Technology), p-IκBα (1:1000, Cell Signaling Technology, 9246), cleaved-Notch1 (1:1000, Cell Signaling Technology, 4147) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh, 1:5000, GeneTex, GTX100118), and finally detected using the Visualizer Kit (Millipore).

Dendrobium officinale was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd. (Jinhua, China), and PMA and LPS were purchased from Sigma (Saint Louis, MI, USA). Recombinant human interleukin-4 (IL-4) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). TNF-α and IL-6 ELISA kits were purchased from Lianke Biological Technology (Hangzhou, China). Cell Counting Kit-8 (CCK-8) Assay Kit was received from MCE (Romulus, NJ, USA). Total Protein Extraction kit was purchased from Beyotime (Shanghai, China). The MiNiBEST Universal RNA Extraction Kit, PrimeScript RT Reagent kit, and SYBR Premix Ex Taq II Kit were purchased from TaKaRa (Dalian, China). Transwell polycarbonate membrane had an 8 μm pore size (Corning City, NY, USA). The primary antibodies E-cadherin, N-cadherin, Vimentin, Caspase-3, Bax, Bcl-2, Ki67, and β-actin were purchased from Proteintech (Wuhan, China). The primary antibodies ARG1, TGM2, and Cleaved-NOTCH1 were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies STAT6, p-STAT6, PPAR-r, JAGGED1, Cleaved-NOTCH1, and NOTCH1 were purchased from Abcam (Cambridge, UK). β-actin was purchased from Proteintech (Wuhan, China). The ECL Plus Western Blotting Detection Kit was purchased from Technology Co., Ltd. (Beijing, China). Anti-CD80-FITC and Anti-CD206-PE were obtained from Thermo Fisher (Waltham, MA, USA).

20 to 50 μg of protein was diluted 1:1 with 2× loading buffer (100 mM Tris–HCL pH 6.8, 20% glycerol, 4% SDS, 200 mM DTT and Bromophenol Blue), and proteins were denatured for 5 min at 95°C. Samples were resolved on a SDS–PAGE following standard procedures.
Gels were transferred onto Nitrocellulose membrane (GE Healthcare) for 1.5 h at 400 mA. The membrane was then blocked with 5% Milk in TBS‐tween buffer (20 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween) for 20 min. Membranes were incubated 30 min/1 h or O/N at 4°C in primary antibodies [Cleaved Notch1 (1:1,000, Cell Signalling #4147); β‐Actin (1:10,000, Proteintech #66009‐1‐Ig); Fbxw7 (1:1,000; Abcam #171961); GFP (1:1,000, Cell Signalling #2956); Cdk1 (1:1,000, Cell Signalling #9116); Cdk2 (1:1,000, Cell Signalling #2546); Cdk4 (1:1,000, Santa Cruz #260); Cdk6 (1:1,000, Santa Cruz #177); Cdk8 (1:1,000, Bethyl #A302‐501A‐T); Cyclin A2 (1:1,000, Cell Signalling #4656); Cyclin B1 (1:1,000, Cell Signalling #4138); Cyclin C (1:1,000, Bethyl #A301‐989A‐M); Cyclin D1 (1:5,000, Abcam #137875); Cyclin E1 (1:1,000, Cell Signalling #4129); Total histone H3 (1:1,000, Cell Signalling #9715); Phospho‐histone H3 (1:1,000, Cell Signalling #3377)].
The membranes were then washed with TBS‐Tween and incubated with the appropriate secondary HRP antibody (Cell Signalling, #7074, #7076). After washing, membranes were developed using ECL solution (Pierce).

All inhibitors were dissolved in DMSO to a concentration of 10 mM and stored under nitrogen. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): Cleaved Notch1 (#4147), PTEN (#9559), pAKT T308 (#2965), pAKT S473 (#9271), pNDRG1 T346 (#5482), pS6RP S235/236 (#2211), PIK3CA (#4249), PIK3CB (#3011), PIK3CG (#5405), TUBULIN (#5346), GAPDH (#3683). PIK3CD (sc-7176), pPRAS40 T246 (44-1100G) and MYC (ab32072) were purchased from Santa Cruz (Dallas, TX, USA), Life Technologies (Grand Island, NY, USA) and Abcam (Cambridge, UK), respectively.