Uvc 500 crosslinker

The E. coli ΔibpB cells transformed with pBAD carrying the gene of IbpB Bpa variant and pSup‐BpaRS‐6TRN plasmid were initially grown at 30 °C to A₆₀₀ = 0.4; induced by arabinose for 2 h to express the IbpB variant protein; washed twice using fresh LB to remove arabinose; incubated at 50 °C for 10 min; and transferred back to 30 °C for a prolonged incubation. Cultures were taken out at indicated time points and immediately transferred to 24‐well plate before being subjected to UV irradiation at 365 nm for 10 min using a Hoefer UVC 500 crosslinker. The cells were lysed, analyzed by 10% Tricine/SDS/PAGE, and then immunoblotted with anti‐His tag monoclonal antibody.

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.

Films (unmodified or modified) were cut to a size that fit into a well plate and washed with DI water. The films were placed into a well plate (Costar^®, Tissue culture-treated well plates, which were used as the control substrate throughout) and sterilised in a Hoefer UVC 500 cross linker for 15 min. After this time the films were turned over with sterilised tweezers and the sterilised side adhered to the well plate with a single drop of Norland optical adhesive 63. The well plate and contents were resterilised (15 min irradiation), PBS (1 mL) was added to each well and the plate stored at 4 °C.
Under sterile conditions, the PBS was removed from the films and 300 μL of DMEM medium, either alone, or with pure FBS or RGD solution (10 μg/mL) as appropriate, added to the wells and left to hydrate for 24 h at 4 °C prior to cell attachment studies.

For ultraviolet light source, UVC 500 Crosslinker (Hoefer Pharmacia Biotech, San Francisco, CA, USA) equipped with three UV-B lamps (type G8T5E, Sankyo Denki, peak wavelength 306 nm) and two UV-A lamps (type TL8WBLB, Philips, peak wavelength 365 nm) were used. Plants were exposed to single radiation episode until a cumulative dose of 1500 mJ cm^-2 was reached (roughly 10 minutes). After 4 days leaves were excised, fresh weight measured (g) and transferred into 50 ml falcon tubes containing 35 ml MilliQ water. The relative electrolyte leakage was measured with a conductance meter (WTW, INOLAB Cond Level 1) and calculated as a ratio between the value obtained after 1 h incubation and the total electrolyte leakage evaluated after autoclaving the samples.

The pYLC‐BamA or pYLC‐BamB plasmid harboring an introduced TAG codon in the bamA or bamB gene was expressed in the LY928, LY928‐∆bamB or LY928‐∆surA cells, respectively. The cells were cultured in the LB medium containing pBpa (200 μm) at 37 °C and grown to the mid‐log phase (with a D₆₀₀ of ~0.8–1.0). Next, the cells were irradiated under the UV light (365 nm) for 10 min in a Hoefer UVC‐500 cross‐linker. Subsequently, the cells irradiated with the UV light were harvested by centrifugation, resuspended in SDS/PAGE loading buffer and boiled for 5 min. The protein samples were resolved by SDS/PAGE and then subjected to blotting analysis.