Phenylmethylsulfonyl fluoride pmsf

An affinity-purified monoclonal anti-FLAG antibody, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), and sodium vanadate were obtained from Sigma Chemical Co. (St. Louis, MO) and anti-pAKT, AKT, phosphorylated ERK1/2, ERK1/2, and RasGRP3 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-Ras and anti-Rap1 antibodies were purchased from BD Biosciences (San Jose, CA). The AKT kinase-dead mutant vector was obtained from Addgene. The H-Ras dominant negative mutant vector was a gift from Professor Raymond Mattingly from Wayne State University (Detroit, MI). The pFLAG-RasGRP3 with an N-terminal FLAG tag was described previously [48 (link)].

The transfected or virus-infected cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA) supplemented with a proteinase inhibitor (20 nM phenylmethylsulfonyl fluoride [PMSF]; Sigma-Aldrich). Cells lysis supernatants were measured and boiled in the buffer for 10 min. Equal amounts of extracts were separated with SDS-PAGE and transferred to methanol-activated polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% skimmed milk in 1× TBST (Tris-buffered saline with 0.05% Tween 20) for 1 h and incubated overnight at 4°C with an antibody against Nat9 (1:12,000), PRRSV Nsp2 (1:2,000), or PRRSV N (1:1,000) or with labeled Abs (1:2,000). This was followed by washing and incubation with HRP-conjugated antibody for 1 h at room temperature. Immunodetection was completed using Pierce enhanced chemiluminescence (ECL) Western blotting substrate (Thermo Fisher Scientific).

For phospho-ERK 1/2 and phospho-Akt determination, cells were serum-starved and treated as described. Cells were then collected and lysed by adding RIPA buffer with sodium orthovanadate (10 µL/mL, Sigma), Phenylmethylsulfonyl fluoride (PMSF) (10 µL/mL), aprotinin (20 µL/mL), and NaF 50 mM (all from Sigma). Cell extracts were resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro blotted onto a nitrocellulose membrane (Trans-Blot Transfer Medium 0.2 µm, BioRad Laboratories, Madrid, Spain). Primary antibodies against Akt (dilution 1:1000), phospho Akt (dilution 1:1000), ERK 1/2 (dilution 1:10,000), phospho ERK 1/2 (dilution 1:1000) (all from Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Heidelberg, Germany; dilution 1:5000) were applied overnight at 4 °C or 4 h at room temperature. Immunoreactive bands were detected with a western-light chemiluminescence detection system (ECL, APBiotech, Little Chalfont, UK) and photographed (HyperfilmECL, APBiotech, IBM, Madrid, Spain). Band density was evaluated with the Image J software.

Ovarian tissues were immediately washed with ice 0.9% saline solution (w/v), weighed and homogenized in a volume of 100 mg tissue/300 μL of lysis buffer (0.1 M Tris, 0.01 M NaCl, 0.025 M EDTA, 1% NP40, 1% Triton X100, Sigma-Aldrich, Milan) supplemented with 2 mM sodium orthovanadate, 0.1 M sodium fluoride (Sigma-Aldrich, Milan), 1:100 mix of protease inhibitors (Sigma-Aldrich, Milan), 1:1000 phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, Milan), using an electric potter at 1600 rpm for 2 min. Samples were mixed for 30 min at 4 °C, centrifuged for 30 min at 13000 rpm at 4 °C and 40 μg of proteins for each samples resolved on SDS-PAGE gel at 15%. Proteins transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-VDR receptor (1:400, Santa-Cruz), anti-ERβ (1:500, Santa-Cruz), anti-cyclin-D1 (1:1000, Euroclone, Milan, Italy). Protein expression was normalized and verified through β-actin detection (1:5000; Sigma-Aldrich) and expressed as a mean ± SD (%).

Purified phosphatidylcholine from soybean lecithin (Phospholipon 90G, CAS-number 97281-47-5) was purchased from Lipoid (Ludwigshafen, Germany). Trizma base, HEPES, Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), glycine, ammonium persulfate, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, 2-mercaptoethanol, Hoechst 33258, and BSA-fraction V were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PVDF membranes, high performance chemiluminescence film, and enhanced chemiluminescence- (ECL-) Plus are from Amersham Biosciences (GE Healthcare, Piscataway, NY, USA). Mini-Protean apparatus for SDS-polyacrylamide electrophoresis, miniature transfer apparatus, acrylamide, bis-acrylamide, and TEMED were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Anti-EGFR (1005) antibody and secondary antibodies conjugated with HRP were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA, USA). Antibodies anti-phospho-mTOR Ser2448, anti-mTOR, anti-p44/42 MAP kinase (ERK 1/2), and anti-phospho-p44/42 MAP kinase Thr202/Tyr204 were from Cell Signaling Technology Inc. (Beverly, MA, USA). Cy3-conjugated secondary antibody against rabbit polyclonal immunoglobulins was from Jackson ImmunoResearch Laboratories, Inc. Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific, Pierce Protein Research Products (Rockford, IL, USA).