Gs 700 imaging densitometer

SDS-PAGE of crude extracellular extracts of B. fibrisolvens 3071 grown in medium with different substrates as described above was performed according to Laemmli [34 (link)] on a slab of 8% polyacrylamide gel in a Mini-Protean Tetra cell system (Bio-Rad) at 110 V for 1.5 h. Protein bands were visualized with Bio-Safe Coomassie R-250 Staining Solution (Bio-Rad) and analysed in a GS-700 Imaging Densitometer (Bio-Rad). Zymograms were prepared according to Flint et al. [35 ] using 0,1% (w/v) carboxy-methyl (CM) xylan as the substrate [31 ] on a slab of 8% polyacrylamide gel under the same conditions as for SDS-PAGE. The gel was washed with 1% (v/w) Triton X100 (Fluka) for 30 min and three times with 25 mM phosphate buffer (pH 7,5) to allow the renaturation of enzymes. The gel was then incubated for 20 min at 39 °C in 25 mM phosphate buffer (pH 7,5) and stained in 0.1% (v/w) Congo Red (Sigma-Aldrich) for 30 min. The highlighted spots indicated xylanase activity against a red background after destaining with l M NaCl.

Total proteins were extracted from cells or tissues using RIPA lysis buffer (Promega, Madison, WI, USA). Proteins were separated on an 8% SDS-PAGE gel and then transferred to PVDF membranes. The membrane was blocked with 5% skim milk at room temperature for 1 h and then incubated with primary antibodies (anti-DLAT, 1:1000 dilution, Abcam, ab51608; anti-SP1, ab101562, 1:1000 dilution, Abcam) at 4 °C overnight, followed by incubating with horseradish peroxidase (HRP) conjugated β-actin secondary antibodies (1:5000) at room temperature for 90 min. The protein bands were scanned and visualized by the GS700 imaging densitometer (Bio-Rad Laboratories) and analyzed by Image Studio software.

SDS-PAGE and Western blot were performed as previously described (13 (link)). Briefly, cells were lysed in RIPA buffer [100 mM tris(hydroxymethyl)aminomethane (Tris)–HCl pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM MgCl₂] in the presence of a complete protease-inhibitor mixture. Protein content was determined by the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA). Cell lysates (30 µg/ml) were loaded onto SDS-PAGE and, after electrophoresis, proteins were transferred onto nitrocellulose membrane (GE Healthcare, Pittsburgh, PA, USA) by means of a Trans-Blot transfer cell (Bio-Rad Laboratories). The membranes were then blocked in 5% nonfat milk and incubated with the appropriate antibodies in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Anti-ERβ mAb (clone CWK-F12 from DSHB) was used as primary Ab. Peroxidase-conjugated goat anti-mouse IgG was used as secondary Ab (Bio-Rad Laboratories) and the reactions were developed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). To ensure the presence of equal amounts of protein, the membranes were reprobed with a rabbit anti-human glyceraldehyde 3-phosphate dehydrogenase Ab (Sigma). Quantification of protein expression was performed by densitometry analysis of the autoradiograms (GS-700 Imaging Densitometer, Bio-Rad Laboratories).

NSCLC tissue or cellular proteins were respectively extracted with cell lysis buffer (Promega, Madison, WI, USA). Thirty micrograms of proteinextraction was separated on an 8% SDS-PAGE gel and then transferred to nitrocellulose membranes. After blockage by milk, the membranes were incubated for 2 h at room temperature with primary antibody (anti-HIF1A, 1:1000 dilution, Abcam, ab51608; anti-LDHA, ab101562, 1:1000 dilution, Abcam). Then, blots were incubated at room temperature for 90 min with horseradish peroxidase (HRP) conjugated beta-actin secondary antibodies (diluted 1:5000). The bands were scanned and visualized by GS700 imaging densitometer (Bio-Rad Laboratories) and analyzed by Image Studio software.

Immunoblotting was as previously described.^{12 (link), 13 (link)} The SK-N-SH cells growing on T75 flasks (n=5 per treatment group) were lysed with radioimmunoprecipitation buffer (20 mm Tris-HCl (pH 7.4), 0.15 mm NaCl, 1% Nonidet P-40 (Sigma, St. Louis, MO, USA), 0.1% SDS (sodium dodecyl sulfate), 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktails (Sigma).
The tissue brain micropunches (300-μm thick) obtained from naive P (n=5), SD (n=5) and P rats infused with pHSVsiTLR4 (n=5) or pHSVsiNCC (n=5) were lysed with CelLytic MT (dialyzable mild detergent, bicine and 150 mm NaCl; Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The total protein was determined by the bicinchoninic assay (BA; Pierce, Rockford, IL, USA). The proteins were resolved by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The blots were exposed to primary antibody overnight (4 °C), followed (1 h; RT) by horseradish peroxidase-labeled secondary antibodies. The detection was with the ECL kit reagents (Amersham Life Science, Pittsburgh, PA, USA) and quantification was by densitometric scanning with a Bio-Rad GS-700 imaging densitometer.