Pvdf transfer membrane

Cell lysates from both control and treated breast cells were prepared by three rounds of freeze‐thawing and vortexing of cell suspensions in a lysis buffer containing 10 mmol/L 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES), pH 7.9, 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol (DTT), 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.5 mg/mL each of leupeptin, antipain, and pepstatin, 0.1 μg/mL chymostatin, 0.3 TIU/mL aprotinin, and 0.5 mg/mL benzamidine. Equal protein amount was fractionated by electrophoresis in sodiumdodecyl sulfate‐polyacrylamide gel (SDS‐PAGE), transferred to PVDF transfer membrane (PerkinElmer, Waltham, MA, USA), stained with Ponceau S solution (Sigma Chemical Co., St. Louis, MO, USA), destained, and immunoblotted with the designated antibodies including β‐actin to ensure equal loading. Anti‐mTOR, anti‐pmTOR (Ser‐2448), anti‐pP70S6K (T‐389), anti‐LC3B I, anti‐LC3B II, anti‐β‐Actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐P70‐S6K antibody was purchased from EMD Millipore Corporation(Billerica, MA, USA). Blots were developed with enhanced chemiluminescece reagent (Pierce ECL, Thermo Scientific, Waltham, MA, USA). Band densitometry was measured by AlphaView imaging software, FluorChem Q system, ProteinSimple, and semiquantitative data were normalized for β‐actin.

For the detection of recombinant hPH20 expression, samples were harvested by centrifugation (9,000 × g for 10 min at 4°C). The supernatant was mixed with 4 × Protein Native PAGE Loading Buffer (TaKaRa, Dalian, China) and heated at 100°C for 10 min. SDS-PAGE was performed by 10% Bis-Tris Protein Gels in MES running buffer at 100 V. Then, SDS-PAGE gel was electroblotted to PVDF transfer membrane (PerkinElmer, Shanghai, China) using a transfer buffer containing 20% methanol, 15.1 g L⁻¹ glycine, and 3.0 g L⁻¹ Tris. Blotted membrane was blocked with QuickBlock™ Blocking Buffer (Beyotime Biotechnology, Shanghai, China) for 1 h. For immunodetection, His-tag Mouse Monoclonal Antibody (Beyotime Biotechnology, Shanghai, China) diluted 1: 1,000 with TBS (2.4 g L⁻¹, pH 7.6 Tris buffer containing 8.0 g L⁻¹ NaCl) was used as a primary antibody. After washing with TBST (2.4 g L⁻¹, pH 7.6 Tris buffer containing 8.0 g L⁻¹ NaCl and 0.1% Tween-20) for 30 min, a 1:1,000 dilution of the secondary antibody, HRP-labeled Goat Anti-Mouse lgG (H + L) (Beyotime Biotechnology, Shanghai, China) was added and incubated at 4°C with gentle shaking for 1 h. After washing with TBST for 30 min, DAB Horseradish Peroxidase Color Development Kit (Beyotime Biotechnology, Shanghai, China) was used to develop the color on the membrane.