Hybond ecl

Protein extracts from Drosophila larval brains were obtained by dissecting 10 larval brains in 0.7% NaCl and homogenizing them in 20 μl of 2X Laemmli buffer. Protein samples were loaded into a 4–20% Mini-PROTEAN TGX precast gel to perform electrophoresis (SDS-PAGE) and blotted using the Trans-Blot^® Turbo™ Transfer System on a nitrocellulose membrane (Hybond ECL, Amersham). Filters were blocked in 5% non-fat dry milk dissolved in 0.1% Tween-20/PBS for 30 min at RT and, then, incubated with anti-Giotto (1:5000; Rabbit; ref. ^{60 (link)}) and anti-γH2Av (1:1000; Mouse; Developmental Studies Hybridoma Bank, IA 52242) overnight at 4 °C. The membranes were then incubated with HRP-conjugated anti-Mouse and anti-Rabbit IgGs secondary antibody (1:5000; Amersham) for 1 h at RT and then washed again 3 times with 0.1%Tween-20/PBS. The chemiluminescent signal was revealed through either SuperSignal™ West Femto or SuperSignal™ West Pico substrate (Thermo Scientific™) using the ChemiDoc scanning system (Bio-Rad). Band intensities were quantified by densitometric analysis using the Image Lab 4.0.1 software (Bio-Rad). WB was repeated independently at least three times.

Cells and each area of the brain tissue were homogenized and lysed for 30 min incubation on ice. The lysates were centrifuged at 14,000 rpm for 15 min. An equal amount of total protein (20 μg) isolated from brain tissues was resolved on an sodium dodecyl sulfate (SDS) 10 or 12 % polyacrylamide gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots were blocked for 1 hr at room temperature with 5 % (w/v) non-fat dried milk in Tris-buffered saline Tween-20 [TBST: 10 mM Tris (pH 8.0) and a 150 mM NaCl solution containing 0.05 % Tween-20]. After a short wash in TBST, membranes were incubated at room temperature with specific. The blot was then incubated with the corresponding conjugated anti-mouse or anti-rabbit antibodies (1:2000, Santa Cruz Biotechnology). Immunoreactive proteins were detected with the enhanced chemiluminescence (ECL) western blotting detection system.

To visualize the interaction of B. cereus C-SpoIISA with the heterologous B. subtilis C-SpoIISA as well as the interaction of B. cereus C-SpoIISA with SpoIISB, we performed Western blotting using the general protocol of Ausubel et al. (1987 ). Briefly, proteins were fractionated by either 12% SDS-PAGE or 16.5% Tricine-SDS-PAGE and transferred onto a nitrocellulose membrane (Hybond ECL; Amersham Bioscience). To prevent non-specific binding, the membrane was treated using 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (v/v). His₆-tagged B. cereus C-SpoIISA was probed with an anti His₆-tag monoclonal antibody (Novagen; catalog no. 70796-3) while S-tagged B. subtilis C-SpoIISA and S-tagged B. cereus SpoIISB were probed with an anti S-tag monoclonal antibody (Novagen; catalog no. 71549-3). Protein interactions were detected using antimouse horseradish peroxidase-conjugated secondary antibodies (Promega; catalog no. W402B).

Urine analysis by immunoblot was performed according to Ferreira and colleagues [21 (link)]. Briefly, concentrated urine samples of each animal were diluted in Tris-buffered saline (TBS) to a final protein concentration of 0.1 µg·µL⁻¹. A volume of 100 µL was slot-blotted onto a nitrocellulose membrane (Hybond^® ECL™, Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK). Nonspecific binding was blocked with 5% (w/v) nonfat dry milk in TBS with Tween 20 (TBST). Each membrane was then incubated with the appropriate primary antibody solution [anti-matrix metalloproteinase (MMP)9 (ab38898) diluted 1:500 and anti-MMP2 (ab37150) diluted 1:200 from Abcam (Cambridge, UK)]. Membranes were then washed, incubated with the secondary antibody diluted 1:10,000 [IRDye^® 800 CW Goat anti-rabbit IgG (926-32211) from LI-COR Biosciences (Lincoln, NE, USA)] and washed again. All antibody solutions were diluted in 5% (w/v) nonfat dry milk in TBST and all incubations were performed for 1 h at room temperature with agitation. Detection was performed with fluorescence according to the manufacturers’ instructions (LI-COR Biosciences, Lincoln, NE, USA). Images were acquired using LI-COR Odyssey^® Scanner (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using Image Studio™ Lite software (LI-COR Biosciences, Lincoln, NE, USA, v5.2.5). Optical densities (OD) values were expressed in arbitrary units.

U266 and U937 cells (1 × 10⁶ cells/mL) were treated with indicated concentrations of SM (25 or 50 µg/mL) for 24 h. Then, the cells were lysed with RIPA buffer (1 M EDTA, 1 mM Na₃VO₄, 1 mM NaF, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholic acid) containing a protease inhibitors cocktail (Amresco, Solon, OH, USA). The protein supernatant was collected and quantified for protein concentration by using an RC DC protein assay kit II (Bio-Rad, Hercules, CA, USA). The proteins (30 µg) were separated via SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes that were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 and incubated with the required antibodies. Primary antibodies, including cleaved PARP (1:1000, 89 kDa), CHOP (1:500, 27 kDa), P-eIF2α (1:500, 38 kDa) (Cell Signaling, Beverly, MA, USA), P-ATF4 (1:500, 39 kDa) (Thermo Fisher, Waltham, MA, USA), P-PERK (1:500, 125 kDa) (Thermo Fisher, Waltham, MA, USA), c-Jun (1:1000, 39 kDa), and β-actin (1:1000, 43 kDa) (Santa Cruz, Dallas, TX, USA), were used at a 1:500~1000 dilution (5% bovine serum albumin) and secondary antibodies at a 1:1000 dilution (5% skim milk) (Santa Cruz, Dallas, TX, USA). After detected protein bands were visualized by Hybond ECL (Amersham Pharmacia, Piscataway, NJ, USA), the blots were scanned.