Polyvinylidene difluoride pvdf membrane

Proteins from retinas of MCMV-infected BALB/c mice, treated or not treated with rapamycin (Selleckchem, Houston, TX), or from retinas of MCMV-infected Atg5^flox/flox; Nestin-Cre mice were extracted on ice by lysis buffer (Roche Diagnostics, Indianapolis, IN). Lysates were clarified at 13,000×g for 10 min at 4 °C and size-fractionated by 10% SDS-PAGE, and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Pittsburgh, PA). The membrane was blocked with 5% nonfat dry milk for 1 h at room temperature, and then was incubated overnight at 4 °C with primary antibody (LC3B; Caspase-3; mammalian target of rapamycin (mTOR); phospho-mTOR; Cell Signaling, Danvers, MA). The next day, after three washes, the membrane was treated with HRP-conjugated secondary antibody 1 h at room temperature. The immune complex was visualized by a chemiluminescence detection system (Thermo Scientific, Waltham, MA) and was exposed to x-ray film. β-actin (Sigma-Aldrich, St. Louis, MO) was used for loading control among each sample. Each experiment was repeated three times.

VSMCs were pretreated with AEPs or AETa (20 μg/ml) and then stimulated during 20 h with AngII (100 nm). Total or fractionated proteins were obtained with a Subcellular Protein Fractionation Kit (#78840; Thermo Fisher Scientific, Waltham, MA, United States). Protein content was determined with Bradford protein assay reagent, and lysates (25 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (Amersham, GE Healthcare, Buckinghamshire, United Kingdom), and probed with antibodies against NOX2 (1:500; #611414; BD Biosciences, San Jose, CA, United States) and NOX4 (1:500; Santa Cruz Biotechnology, Dallas, TX, United States). Detection was accomplished using horseradish peroxidase-coupled anti-rabbit or anti-mouse (1:2,000; The Jackson Laboratory, Bar Harbor, ME, United States) IgG antibody for 1 h at room temperature. Signal was detected using BioLumina (Kalium Tech, Buenos Aires, Argentina) detection system. Immunoblot signals were quantified by NIH ImageJ using β-actin expression as loading control.

The whole cell lysates from breast cancer cell lines (MCF 10A, MCF-7, ZR-75-1, T-47D, SKBR3, MDA-MB-231 and MDA-MB-468) were obtained by using RIPA buffer (500 mM NaCl, 5 mM MgCl2, 1% Na deoxycholate, 20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EDTA, 100 mM EGTA, 0.1% NP40, 1% Triton X-100, 0.1 M Na3VO4, 1X Protease inhibitor, 1X Phosphatase Inhibitor). Protein concentration was calculated by the Bradford protein assay method. Total protein extracts (40 μg) were electrophoresed in 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, Chalfont, UK). Blots were incubated with 5% skimmed milk (MP Biomedicals, India) for 1 h of blocking. Then the membrane was cut prior to incubation with primary antibodies and kept at 4°C overnight with gentle shaking (details of all antibodies and reagents are provided in the (Supplementary Table 1)). The membrane was then washed with 1X TBS-T and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h. After washing, the blots were developed using Western Blot Chemiluminescence HRP Substrate (Takara Bio Inc.) in Chemidoc XRS+ molecular 228 imager (Bio-Rad, Hercules, CA, USA). The images were quantified using Image J software (NIH, Bethesda, MD, USA).

The algU gene region containing the 500 bp native promoter was cloned by PCR with the designed primers (algU-flag-F/R) (Table S2). The algU-flag-R primer was added with the flag tag sequence. The algU-flag PCR amplicons were cloned into the pGD926 vector with recombinase, and the product was named pGalgU-flag. pGalgU-flag was then introduced into P. stutzeri wild-type A1501 or selected mutants by triparental mating with the helper vector pRK2013. Western blotting was performed using protein extracts of bacterial cells grown for 10 h in K medium containing 50 mM lactate and 6 mM NH₄Cl. Five micrograms of total protein were loaded and separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined using the Bio-Rad protein assay reagent kit. The separated protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Piscataway, NJ, USA). The membrane was incubated with a monoclonal antibody against the flag peptide (Sigma-Aldrich Co. LLC, Saint Louis, MO, USA) for 12 h at 4 °C and then washed three times in TBS/Tween buffer before being incubated with an anti-mouse secondary antibody (Sangon Biotech Co., Ltd., Shanghai, China) for 2 h. Detection was performed using an HRP-DAB chemistry kit (Tiangen Biotech Co., Ltd., Beijing, China).

Penicillin, streptomycin, sodium pyruvate, L-glutamine and fetal calf serum (FCS) were purchased from Invitrogen-Gibco (Paisley, UK). Geniticin, aprotinin, pepstatin and phenylmethylsulfonyl fluoride (PMSF) were from Sigma-Aldrich (Taufkirchen, Germany). Leupeptin was purchased from MP Biomedicals (Illkirch, France). Sodium orthovanadate was obtained from Merck (Darmstadt, Germany). DMSO and JAK inhibitor I (JI-1) were from Calbiochem (Merck, Darmstadt, Germany). Ficoll Paque and the polyvinylidene difluoride (PVDF) membrane were purchased from GE Healthcare (Munich, Germany). The enhanced chemiluminescence kit was obtained from Thermo Fisher Scientific (Bonn, Germany). Protein concentrations were determined with the Bio-Rad reagent (Bio-Rad, München, Germany).