Lightshift chemiluminescent emsa kit

To test the binding of FTZ-F1 to regulatory sequences in the CPLCG5 promoter, an EMSA experiment was performed using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Waltham, MA, USA). The potential FTZ-F1-binding sequence from CPLCG5 (− 870 to − 864) was used as a probe and labeled with biotin at the 5′-end. The sequences of the cold probes were the same as those of the labeled probes. Labeled probes and mutant probes were used as competitors for each other. The probes were synthesized by Invitrogen (Shanghai, China). A nuclear extract was obtained by using the NE-PER® Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Membranes were made using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The probe and nuclear protein were loaded onto 6.5% polyacrylamide gel and electrophoresed at 100 V for 60 min.

EMSA was carried out using the LightShift® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. NE-PER® Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Inc.) was used to prepare nuclear extracts from H9c2 and HEK-293 cells, the concentrations of nuclear extract were measured by Bradford protein assay. Double-stranded oligonucleotide fragments (30 bp) containing DSVs were biotinylated and used as probes (Table 1). Nuclear extracts (3.0 μg) and probes (0.2 pmol) were incubated for 20 min at room temperature, separated on a native 6% polyacrylamide gel at 100 V for 90 min, and then transferred onto a nylon membrane (Thermo Fisher Scientific, Inc.) at 380 mA for 30 min. UV Stratalinker 1800 (Stratagene, Santa Clara, CA, USA) was used to cross-link oligonucleotides onto the nylon membrane, and the LightShift® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc.) was used for chemiluminescence detection.

To examine the effects of TFEB gene regulatory variants on the binding sites for transcription factors, electrophoretic mobility shift assay (EMSA) was conducted using the LightShift^® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States). Biotinylated double-stranded oligonucleotides (30 bp) containing regulatory variants were used as probes. Nuclear extracts from HEK-293 and H9c2 cells were prepared using NE-PER^® Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States). Protein concentrations were determined using the Bradford protein assay. DNA-protein binding reactions were conducted for 20 min at room temperature with equal amounts of probes (0.2 pMol) and nuclear extracts (3.0 μg). The reaction mixtures were subsequently separated on a 6% polyacrylamide gel and transferred onto a nylon membrane (Thermo Fisher Scientific, Inc., Waltham, MA, United States). The oligonucleotides were cross-linked to the membrane using the UV Stratalinker 1800 (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, United States) and were detected by chemiluminescence using the LightShift^® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States).

The DNA EMSA experiment was performed using a LightShift® Chemiluminescent EMSA Kit (Thermo Scientific) by following the manufacturer’s protocol. The HIF-1α and Jumu proteins were purified from the HEK293T cells transiently transfected with HIF-1α and Jumu plasmids using anti-HA and anti-Flag conjugated agarose (Sigma-Aldrich). Each DNA EMSA reaction mixture was set up in a final volume of 20 μl containing the annealed double-stranded DNA probe (20 nM) and HIF-1α /Jumu protein (500 ng) in the binding buffer of the LightShift® Chemiluminescent EMSA Kit (Thermo Scientific). The biotin-labeled probes used for the DNA EMSA assay are listed (Additional file 2: Table S18).

The Thermo Scientific LightShift Chemiluminescent EMSA Kit (Thermo Scientific Pierce) was used to detect the interactions of NF-κB and miR-99a promoter. A 20 bp of probe covering the miR-99a promoter sequence between −1639 and −1658 was used for EMSA. The sequence of the wild-type probe is 5′-TTT TAG GGA AAA ACT TAA AA-3′, and the sequence of mutant-type probe is 5′-TTT TAT TTA AAG GCC TAA AA-3′ (mutations are underlined and italic). A positive NF-κB probe with the sequence of 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ was used. The above three DNA fragments were biotin-labeled using biotin 3′ end DNA labeling kit (Viagene Biotech Inc.). The binding reaction was performed by adding 4 μg of nuclear extracts, 20 fmol of biotin end-labeled DNA (or unlabeled DNA for competitive assay), and 1 μg/μL poly(dI-dC) to the end volume of 15 μL. After incubation for 20 min at room temperature, the reaction is then subjected to gel electrophoresis on a 6% native polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA was detected using the Streptavidin-Horseradish Peroxidase Conjugate and the Chemiluminescent Substrate.

Nuclear extracts were prepared using a nuclear and cytoplasmic extraction kit (CWBIO, China). The single-strand oligonucleotides was labeled with biotin on the 3′ end DNA, annealed to form double-stranded oligonucleotides containing the tested NF-κB sequences from the pFcRn promoter: pFcRn κB-1286 (5′-AAAAATGGGA GTTTCCATTTCCG-3′), pFcRn κB-1128 (5′-GTAGCCTGGGAACTTCCAGAT GCC-3′), pFcRn κB-642 (5′-CCAGAAGAGGCAAATTCCTAGAGAC-3′) and pFcRn κB-563 (5′-AAGGGGTACGGGGTCTCCTTGGGG-3′). For competition assays, a 50-fold excess of nonlabeled oligonucleotide was incubated during the preincubation time. For the supershift assays, 1 μg anti-p65 monoclonal antibody directed against NF-κB p65 was preincubated with the nuclear extracts. The complexes were run on a 5% native polyacrylamide gel. EMSA experiments were performed according to the LightShift chemiluminescent EMSA kit (Thermo Scientific Pierce, USA).