Ab34682

The liver tissue and HepG2 cells were lysed by RIPA lysis solution (Beyotime, China) to extract the protein. The protein concentration was detected by BCA reaction kit. After quantitative analysis, the total protein was denatured in this study. SDS-Page gel was used for electrophoresis, electrophoresis apparatus (Bio-RAD, USA) was adjusted to 120 V for electrophoresis, PVDF membrane (Millipore, USA) was used for membrane transfer, and skim milk (Sigma, USA) for blocking. Primary antibodies (Abcam, UK): Sirt1 (1:1000; ab189494), optic atrophy 1 (Opa1, 1:1000; ab157457), mitofusin 2 (Mfn2, 1:1000; ab124773), Drp1 (1:1000; ab184247), NRF1 (1:1000; ab34682), mitochondrial transcription factor A (TFAM, 1:1000; ab252432; Abcam; UK), Bcl-2 (1:2000; ab182858), cleaved-caspase 3 (1:500; ab2302), BCL2-associated X protein (Bax, 1:1000; ab32503), cleaved-caspase 9 (1:2000; ab32539) and GAPDH (1:2500; ab9485) were then added overnight to incubate. The next day, goat anti-rabbit antibody (1:2000; ab288151) was incubated for 1 h with slow shaking at 25 °C. The immunoreactive bands were visualized by intensive chemiluminescent reagent. The gray value was analyzed by ImageJ software.

L02, HepG2 and Hep3B cells were collected and lysed with RIPA buffer (Sigma-Aldrich, USA). Lysates were quantified using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. 30 µg extracted protein were loaded into each lane of the SDS-PAGE gel for separation. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA), followed by blocking with 5% skim milk for 45 min at room temperature. Then, the membranes were incubated with primary antibodies at 4 °C overnight. Goat anti-rabbit IgG antibody (1:2000 dilution; ab7090, abcam, USA) was used as the secondary antibody for 2 h incubation at room temperature. GAPDH was selected as an internal control. Immunoreactive bands were visualized by ECL reagent (Amersham Biosciences UK Ltd., UK) and quantified by densitometry using ImageJ software (National Institutes of Health, USA). Primary antibodies were anti-RHOB (1:1000 dilution; ab277779, abcam, USA), anti-NRF1 (1:1000 dilution; ab34682, abcam, USA), anti-SPIDR (1:1000 dilution; HPA041582, Sigma-Aldrich, USA) and anti-GAPDH (1:1000 dilution; ab8245, abcam, USA).

A ChIP assay was performed using an EpiQuik TM Chromatin Immunoprecipitation Kit (Epigentek) following the protocol provided by the supplier. 2 × 10⁶ Jurkat-HBZ cells and control cells were used in each assay. The sonicated samples were immunoprecipitated with the NRF-1 antibody (ab34682, Abcam) or normal rabbit IgG (sc-2027 X, Santa Cruz) overnight at 4 °C. Protein-DNA complexes were de-crosslinked at 65 °C for 6 h. Primers used for the quantification of the target fragments by real-time PCR were 5′-TGCCGCCAGGGTTTGAA-3′ and 5′-CTGAGGCGCACAGAACCAAC-3′, which were constructed to contain the NRF-1-binding sequence located in −41/−30 bp of the TDP1-gene promoter. Individual PCRs were carried out in triplicate, and mean Ct values were collected.