Western blot hyper hrp substrate

Immunoblot analysis was carried out using the indicated antibodies as described previously, with modifications (33 (link), 50 (link)). Antibody-reactive proteins were detected using ECL Start (Cytiva) and Western blot hyper HRP substrate (Takara Bio) followed by exposure to X-ray film (Fujifilm) and the Fusion Solo 7S Edge chemiluminescence imaging system (Vilber-Lourmat), respectively.

Protein extracts from tobacco leaves were prepared by grinding them in 5 volumes of buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM DTT, and Complete protease inhibitor cocktail (Roche Applied Science)]. Supernatants were cleared by centrifugation at 21,500×g for 15 min at 4 °C, and concentration of the protein extracts was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, USA) with bovine gamma-globulin as the standard.
For immunoblotting analyses, proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck, Germany). After blocking with 5% nonfat dry milk, membranes were probed with 0.1 μg/ml anti-EAS antibody diluted with Western BLoT Immuno Booster (Takara, Japan) at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibody diluted with 1% nonfat dry milk at room temperature for 1 h. The antigen-antibody complexes were visualized using Western BLoT Hyper HRP Substrate (Takara, Japan).

The immunoblot analysis was performed as described previously [44 (link)], using SuperSignal West Pico Substrate (Pierce, Rockford, IL, USA) and Western BLoT Hyper HRP Substrate (Takara Bio Inc., Shiga, Japan) according to the manufacturers’ instructions. Images were analysed using UN-SCAN-Itgel Automated Digitizing System software (Version 5.1 for Windows, Silk Scientific Inc., Orem, UT, USA). The antibodies to the following chemicals were used: eIF4E, p-eIF4E (Ser209), MNK1, MNK2, vimentin, N-cadherin, and E-cadherin. β-actin was used as a loading control.

B. subtilis strains were grown in Soytone medium at 37 °C with shaking. Bacterial cells were harvested when the cultures reached 50 units at OD₆₀₀ and washed three times with cold lysis buffer containing 20 mM Tris/HCl (pH 8), 10 mM NaCl, 10 mM EGTA, 5 mM EDTA, and 50 mM 2-mercaptoethanol, then stored at −80 °C. The cells were suspended in 10 mL lysis buffer containing 100 μL Halt Protease Inhibitor Single-Use Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and 35 μL 2-mercaptoethanol, then disrupted by three passages at 120 bar in Avestin Emulsiflex B15 cell disruptor (ATA Scientific, New South Wales, Australia). Following centrifugation, supernatants were mixed with 1.0% (w/w) protamine sulfate to precipitate nucleic acids. After further centrifugation, supernatants were subjected to 12% PAGE and the proteins separated in the gel were transferred to an iBlot PVDF membrane (Thermo Fisher Scientific, Waltham, MA, USA), where they were subsequently reacted with 2,000-times diluted THE^TM His-tag antibodies (Genescript, Piscataway, NJ, USA) and thereafter with 20,000-times diluted secondary anti-mouse IgG antibodies (Sigma Aldrich, St. Louis, MO, USA). Proteins were revealed using Western BLoT Hyper HRP Substrate (Takara Bio, Shiga, Japan) and visualized with ChemiDoc (Bio-Rad).

Immunoblot analysis was performed as described previously [22 (link)], using SuperSignal West Pico Substrate (Pierce, Rockford, IL, USA) and Western BLoT Hyper HRP Substrate (Takara Bio Inc) according to the manufacturers’ instructions. The images were analyzed using UN-SCAN-Itgel Automated Digitizing System software (Version 5.1 for Windows, Silk Scientific Inc., Orem, UT, USA). The antibodies to the following chemicals were used: 4EBP1, p4EBP1 (The70, Thr37/46, and Ser65), S6K, pS6K (Ser371), ribosomal protein S6 (S6RP), pS6RP (Ser240/244), glycogen synthase (GS), pGS (Ser641), Akt, pAkt (Ser473), GSK-3β and GSK-3α. These antibodies were obtained from Cell Signaling Technology Japan (Osaka, Japan). β-actin was used as a loading control and anti-β-actin was obtained from Abcam Inc. (Cambridge, MA, USA).

hAECs were lysed with PROPREP (iNtRON Biotechnology, MA, USA) supplemented with Phosphatase Inhibitor Cocktail (EDTA free) (Nakarai tesque, Japan). Protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS–PAGE; Bio-Rad Laboratories, CA, USA) followed by a Western blot assay. EzBlockChemi (ATTO, Japan) was used for blocking, and Western BLoT Immuno Booster (Takara, Japan) was used for the reaction. The primary antibodies were as follows: Smad1, Smad2/3, phospho-Smad1 (Ser463/465)/5 (Ser463/465)/9 (Ser465/467), and phospho-Smad2 (Ser465/467)/3 (Ser423/425) (Cell Signaling Technology, MA, USA) at a 1:1,000 concentration, and GAPDH (GeneTex, CA, USA) at a 1:10,000 concentration. The secondary antibodies were peroxidase AffiniPure goat anti-mouse IgG and anti-rabbit IgG (Jackson ImmunoResearch Laboratory, PA, USA) at a 1:20,000 concentration. Western blot images were developed with Western BLoT Hyper HRP Substrate (Takara, Japan) and taken with a LumiCube System (Liponics, Japan).