Trans blot sd semi dry electrophoretic transfer cell

Total RNA was isolated from the tissue samples of the control plants and 340 mM NaCl-treated (9 h) S. maritima using miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions. For the Northern blot analysis, 10 μg of total RNA was resolved on a 15 % urea-PAGE gel. The electrophoresed RNA was transferred onto a nylon membrane using a Trans-Blot^® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The blot was air dried and UV cross-linked at 150 mJ using a UV cross-linker (Hoefer™ UVC 500 Crosslinker). Probes designed from DNA oligonucleotides complementary to the miRNA sequences (Additional file 2) were end-labeled with [γ-³²P]dATP using T4 Polynucleotide Kinase (Fermentas) according to the manufacturer’s instructions. The membrane was pre-hybridized for 1 h with hybridization buffer (Sigma), and then the labeled probe was added and allowed to hybridize for 16 h at 37 °C. After hybridization, the membrane was washed with 2X SSC and 0.1 % SDS at 32 °C for 15 min and 1X SSC and 0.1 % SDS for 15 min at 37 °C. The membrane was air dried and then exposed to X-ray film. When required, the membrane was stripped, re-exposed to the X-ray film to ensure complete signal removal and reused for a second hybridization. A DNA oligonucleotide complementary to U6 snRNA was used as a probe to detect the U6 snRNA for use as an internal control.

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL, USA). Cell lysates were resolved by SDS–PAGE (10%) and proteins were transferred to nitrocellulose membranes (ECL Hybond^TM; Amersham, UK) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Marne-la-Coquette, France). Then, membranes were subjected to immunoblot analysis using primary antibodies and appropriate peroxidase-conjugated secondary antibodies. Primary antibodies (dilution 1/1000) were from Cell Signaling Technology (CST, Danvers, MA, USA) and directed against: GAPDH, acetylated-CBP (K1535)/p300 (K1499), STAT1 and phosphorylated-STAT1 (Y701). Antibody-bound proteins were detected using an enhanced chemiluminescence kit (West Pico ECL, Thermo Fischer) and a luminescent image analyzer LAS-4000 (Fujifilm, Tokyo, Japan). Blot and image analysis were performed with Multi Gauge^® (Fujifilm) and ImageJ^® software. Results shown are representative of at least three independent experiments.

MSCs were lysed with lysis buffer including 1 % phenylmethane sulfonylfluoride (PMSF) and 1 % phosphatase inhibitors and the protein was extracted. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The protein sample extracted from MSCs was separated by 10 % SDS-PAGE (for Pim-1 and β-actin) and transferred to a 0.45 mm nitrocellulose membrane (Millipore, Billerica, MA, USA) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, California, USA). Milk 5 % (w/v) in 0.1 % Tween 20/PBS was used to block the membranes for 1 h. The membranes were then incubated with rabbit polyclonal antibody against Pim-1 (1:100; Santa Cruz, Dallas, Texas, USA) and β-actin (1:1000; Beyotime Biotechnology) overnight and blotted with a HRP-linked secondary antibody (1:1000; Beyotime Biotechnology) for 2 h. Afterwards, the protein band could be visualized via enhanced chemiluminescence and analyzed by the Scion Image Software (Scion, Frederich, MD, USA).

Cassava leaves were harvested and ground by liquid nitrogen and phosphate buffer solution (pH 7.4). The extracted solution was centrifuged at 12,000 rpm and 4 ℃ for 10 min. The supernatant was boiled with SDS-PAGE sample loading buffer (P0015, Biyotime, Shanghai, China) for 5 min. After centrifugation for 10 min, the supernatant could be used in Western blot. The Western blot assay was performed according to a previous study [53 (link)]. Briefly, the protein samples were loaded into 12% polyacrylamide gel and separated by electrophoresis. The polyacrylamide gel was transferred to a PVDF membrane (475855-1R, Millpore, Massachusetts, America) by a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (1703940, Bio-rad, Hercules, America). Then, the PVDF membrane was blocked and incubated in 5% skim milk with anti-GFP antibody (AG281, Biyotime, Haimen, China).

The cells were incubated with CytoBuster^TM Protein Extraction Reagent (Novagen, Darmstadt, Germany), protease inhibitor cocktail (Sigma-Aldrich, MO, USA) and phosphatase inhibitor cocktail 2 (Sigma-Aldrich) for 30 min at 4 °C. The cell extracts were then purified by centrifugation at 12,000 rpm for 15 min at 4 °C, and protein concentrations were determined by Coomassie Plus^TM Protein Assay Reagent (Thermo Fisher Scientific). Protein samples (15 μg) were mixed with Tricine sample buffer (Protech Technology, Taipei, Taiwan), heated for 5 min at 95 °C, resolved on a 4–12% Bis/Tris NuPAGE gel (Invitrogen, Carlsbad, CA, USA) and transferred to a Nitrocellulose blotting membrane (Amersham Biosciences, Piscataway, NJ, USA) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes was subsequently blocked with 5% Difco^TM Skim Milk (BD Biosciences, Franklin Lakes, NJ, USA) in PBS for 1 h, hybridized with the indicated Ab and then incubated with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG or goat anti-rabbit IgG (Millipore, Billerica, MA, USA) for 1 h before being incubated with Immobilon^TM Western Chemiluminescent HRP Substrate (Millipore). The immuno-reactive signals were stripped by Restore^TM Western Blot Stripping Buffer (Thermo Fisher Scientific) before the protein of interest was re-probed.