Biodyne b nylon membrane

For the EMSA, a lightshift chemiluminescent EMSA kit (Thermo Scientific, Rockford, IL) was used, and the manufacturer’s protocol was followed. We designed appropriate oligonucleotide pairs that included the putative binding regions for each pump gene. DNA labeling was performed using a Biotin 3´ end DNA labeling kit (Thermo Scientific). The DNA probes were prepared through PCR amplification. DNA (20 fmol) and His-BaeR protein (1 μg) were mixed in binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, and 1 mM DTT) and poly deoxyinosinic-deoxycytidylic acid (poly (dI-dC)) (50 ng/μL). The reaction mixtures (20 μl) were loaded onto a 5% polyacrylamide gel in 0.5 X TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA at pH 8.3). The DNA-protein complexes were separated at 100 V for 2 hours in 0.5 X TBE buffer and then transferred to a Biodyne B nylon membrane (Pall Corporation, Port Washington, NY). Crosslinking and detection of the His-labeled DNA-protein complexes were performed using a UV lamp and chemiluminescence, respectively.

The gel shift assay was performed using the EMSA kit purchased from Panomics (Affymetrix). HK-2 cells were grown in 60-mm dishes at a density of 250 cells/mm² and cultured for 48 h. After treatment, HK-2 cells were separated into nuclear and post-nuclear fractions. Nuclear protein (3 µg) was incubated with 10 ng DNA probe (biotin-labeled binding sequence to transcription factor) and 1 µg poly d(I-C) with binding buffer for 30 min at 15 °C in a thermal cycler (Takara Bio, Shiga, Japan). For the competition assay, 1,320 ng cold DNA probe was added. The protein-bound probe was electrophoresed on a 5.0% (w/v) TBE (Tris borate EDTA)-polyacrylamide gel in 0.5 × TBE buffer at 4 °C and then transferred to a Biodyne® B nylon membrane (Pall Corporation, Port Washington, NY, USA) in 0.5 × TBE buffer. The membrane was fixed by UV crosslinking (CL-1000 Ultraviolet Crosslinker; UVP, Upland, CA, USA) with 120 mJ/cm². The membrane was blocked and probed with Streptavidin-HRP. The chemiluminescence images were taken using a LAS-3000 device.

The gel shift assay was performed using the EMSA kit purchased from Panomics. Nuclear extractions from control and Cd-treated HK-2 cells were used for the assay. The protein concentration of nuclei was measured using the BCA protein assay kit. Nuclear protein (3 μg) was incubated with 10 ng DNA probe (biotin-labeled binding sequence to transcription factor) and 1 μg poly d(I-C) with binding buffer for 30 min at 15 °C in a thermal cycler (Takara Bio). For the competition assay, 1,320 ng cold DNA probe was added. The protein-bound probe was electrophoresed on a 5.0% (w/v) TBE [Tris borate-ethylenediaminetetraacetic acid (EDTA)]-polyacrylamide gel in 0.5 × TBE buffer at 4 °C and then transferred to a Biodyne^® B nylon membrane (Pall corporation, Port Washington, NY, USA) in 0.5 × TBE buffer. The membrane was fixed by UV crosslinking (CL-1000 Ultraviolet Crosslinker; UVP, Upland, CA, USA) with 120 mJ/cm². The membrane was blocked and probed with Streptavidin-HRP. The chemiluminescence images were taken using a LAS-3000 device.

EMSA was used to detect whether PpBL could directly bind to the cis-acting element of PpACO1 and PpACS1 promoter in vitro. The CDS sequence of PpBL was cloned and inserted into Pgex-6p-1 vector, and the recombinant vector was transformed into Rosetta (DE3) for induction expression. Single-stranded oligonucleotides labeled with or without biotin were used as the probes. The probes were incubated with the nuclear extract at room temperature for 30 min. The entire reaction mixture was run on a non-denaturing 0.5 × TBE 6% polyacrylamide gel for 1 h at 60 V at 4°C and then transferred onto Biodyne® B nylon membranes (Pall Corporation, NY, USA). Signals were visualized with reagents included in the kit and ChemiDoc XRS (Bio-Rad Laboratories, CA, USA) [78 ].

The sequences of the synthetic double-stranded biotinylated oligonucleotides were provided in Supplementary Table S4. The probes were incubated with the nuclear proteins from HEK 293T cells at room temperature for 30 min. For supershift assays, 10 μl of anti-EWS (catalog no. 11910S; Cell Signaling Technology) antibody was incubated with nuclear proteins from HEK 293T cells for 1 h before adding the relevant labeled probe. The entire reaction mixture was run on a non-denaturing 0.5×TBE 6% polyacrylamide gel for 1h at 60 V at 4°C, transferred onto Biodyne® B nylon membranes (Pall Corporation), and crosslinked at 120 × 100 μJ/cm². Signals were visualized with reagents included in the kit and ChemiDoc XRS (Bio-Rad Laboratories, USA).

HEK293T cells were transfected with FLAG-tagged WT or bloto Zfp335
(cloned as described) and nuclear extracts prepared using a modified Dignam protocol.
Relative protein amounts were determined by Western blot using anti-Zfp335
(A300-797A). Gel mobility shift assays were performed as described (Lo et al., 1991 (link)). Briefly, nuclear extracts
were mixed with 10 mM HEPES pH 7.9, 0.1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.1 mM
PMSF, 0.08 mg/ml BSA, 0.04 mg/ml poly dI/dC, 0.4 mM ZnCl₂, and 1ʹ mM
biotin-labeled probe and incubated at room temperature for 20 min. For experiments
using competitor oligos, 20- (Figure 7F) or
50-fold (Figure 7G) molar excess of unlabeled
probe was pre-incubated with nuclear extracts on ice for 30 min before adding
biotinylated probe. Samples were loaded on a 4% polyacrylamide gel with 3.5% glycerol
in 0.25X TBE and run at constant voltage in 0.25X TBE running buffer, then
transferred to Biodyne B nylon membranes (Pall Corporation, Port Washington, NY) in
0.5X TBE at 380 mA for 1 hr. Signal was detected with LightShift Chemiluminescent
Nucleic Acid Detection Module kit (Thermo Scientific, Waltham, MA). All probes were
derived from synthetic double-stranded 5ʹ-biotinylated oligonucleotides, listed in
Supplementary file 5(Integrated DNA Technologies, San Jose, CA).