Ndufa4l2

Manufactured by Abcam

Sourced in United Kingdom

NDUFA4L2 is a protein-coding gene that is involved in the electron transport chain within the mitochondria. It plays a role in the assembly and function of complex I, which is the first step in the mitochondrial respiratory chain. The NDUFA4L2 gene provides instructions for producing a subunit of complex I.

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Lab products found in correlation

Gapdh, by Abcam (2 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Antibiotic antimycotic agent, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Sc 37007, by Santa Cruz Biotechnology (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Ndufa4l2, by Proteintech (1 mentions) Flag tag (flag), by GenScript (1 mentions) Ubiquitin, by Santa Cruz Biotechnology (1 mentions) Cytochrome c, by Cell Signaling Technology (1 mentions) Odyssey clx imaging system, by LI COR (1 mentions) Sqstm1 p62, by Cell Signaling Technology (1 mentions) Cox 2, by Santa Cruz Biotechnology (1 mentions) Phospho akt, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)

5 protocols using ndufa4l2

Silencing ELK1 in ccRCC and ACHN Cell Lines

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Human ccRCC 786-O (Williams et al., 1978 (link)) and ACHN (Kochevar, 1990 (link)) cell lines (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown in RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) (both from Gibco, Waltham, MA, USA), respectively. These media contain 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic agent (Life Technologies, Inc., Grand Island, NY, USA). Cells were grown at 37 °C and 5% CO₂.
Small interfering RNA (siRNA) duplexes pools directed against ELK1 (sc-35290) and control scrambled RNAi (sc-37007) were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were grown to 80% confluency in 35-mm dishes, transfected with 2 µl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and mixed with 30 pmol of the synthetic ELK1 siRNA pool. The cells were then harvested and analyzed after 48 h of transfection.
The primary antibodies specific for ELK1 was bought from Santa Cruz, NDUFA4L2 and GAPDH antibodies were purchased from Abcam (Cambridge, MA, USA).

Wang L., Peng Z., Wang K., Qi Y., Yang Y., Zhang Y., An X., Luo S, & Zheng J. (2017). NDUFA4L2 is associated with clear cell renal cell carcinoma malignancy and is regulated by ELK1. PeerJ, 5, e4065.

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Quantitative Protein Analysis via Western Blot

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Western blotting was performed as previously described.^{8 (link)} Primary antibodies were used (1:1,000 in 5% milk): NDUFA4L2 (Abcam ab74138, rabbit polyclonal), NDUFA4L2 (Proteintech 66050-1-Ig, mouse monoclonal), FLAG (GenScript, A00187S, mouse monoclonal). Secondary antibodies: anti-Rabbit IgG (Jackson, 711–135-052, 1:10,000 diluted in 5% milk), anti-Mouse IgG (Jackson, 715–035-150, 1:10,000 diluted in 5% milk).

Kubala J.M., Laursen K.B., Schreiner R., Williams R.M., van der Mijn J.C., Crowley M.J., Mongan N.P., Nanus D.M., Heller D.A, & Gudas L.J. (2023). NDUFA4L2 reduces mitochondrial respiration resulting in defective lysosomal trafficking in clear cell renal cell carcinoma. Cancer Biology & Therapy, 24(1), 2170669.

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Western Blot Analysis of Cellular Proteins

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NRVCs were washed with PBS and then scraped in RIPA lysis buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS] supplemented with fresh protease inhibitor cocktail (Sigma-Aldrich). Equal aliquots of cell lysates were loaded onto 10% or 12% SDS-polyacrylamide gels for electrophoresis using Bio-Rad’s western blotting system. The gels were transferred onto wet 0.1 μm nitrocellulose membranes (LI-COR, Inc., Lincoln, NE). Membranes were probed with primary antibodies (3 hr at RT) and washed with PBST (1 x, 5 min) containing 0.5% Tween 20 and PBS (3 x, 5 min ea.). Blots were then probed with appropriate secondary antibodies (1 hr at RT) followed by washing with PBST and PBS. Blots were scanned with ODYSSEY^® CLx Imaging system (LI-COR, Inc., Lincoln, NE). Protein band intensities were quantified using Image Studio Software. The following antibodies were used for western blotting: BAG3 (Proteintech, 10599-1-AP), SQSTM1/p62 (Cell Signaling Technology, 5114), LC3 (Sigma, L8918), GAPDH (Santa Cruz, sc-32233), Cox-2 (Santa Cruz, sc-1745), Cytochrome c (Cell Signaling Technology, 4272), BAX (Santa Cruz, sc-493), Ubiquitin (Santa Cruz, sc-8017), NDUFA4L2 (Abcam, ab74138), Phospho-AKT (Cell Signaling Technology, 9271), ATG7 (Cell Signaling Technology, 2631) and Tat (NIH AIDS Reagent Program, R705).

Tahrir F.G., Shanmughapriya S., Ahooyi T.M., Knezevic T., Gupta M.K., Kontos C.D., McClung J.M., Madesh M., Gordon J., Feldman A.M., Cheung J.Y, & Khalili K. (2017). Dysregulation of Mitochondrial Bioenergetics and Quality Control by HIV-1 Tat in Cardiomyocytes. Journal of cellular physiology, 233(2), 748-758.

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Confirming NDUFA4L2 and NXPH4 Interactions

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Western blot analyses were performed as previously described [8 (link)]. Briefly, protein samples lysed from bladder cancer cells or tissues were denatured, electrophoretically separated in 10% polyacrylamide gels, and transferred to poly (vinylidene difluoride) membranes (Millipore, Burlington, MA, USA). The blots were then probed with primary antibodies against NDUFA4L2 (1:200, Abcam, Cambridge, UK), NXPH4 (1:1000, Abmart, Berkeley Heights, NJ, USA), and GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA). The grayscale of the indicated protein was measured with the software Image J (NIH Image, Maryland, USA). For co-immunoprecipitation, the collected cells were mixed by pipetting with a pre-chilled native lysate NETN (20 mM pH8.0 Tris HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) and supplemented with protease inhibitors. The supernatant was incubated with antibodies overnight at 4 °C, followed by protein A/G plus agarose beads for 4 h at 4 °C. The immunoprecipitates were collected by centrifuging the samples and washing 5 times with lysis buffer. Western blotting is usually performed in the order of Co-IP group, Input1, Marker, IP group, and Input2.

Wang D., Zhang P., Liu Z., Xing Y, & Xiao Y. (2022). NXPH4 Promotes Gemcitabine Resistance in Bladder Cancer by Enhancing Reactive Oxygen Species and Glycolysis Activation through Modulating NDUFA4L2. Cancers, 14(15), 3782.

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Quantitative Protein Expression Analysis

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Total proteins were separated by SDS-PAGE after denaturation and transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with rabbit anti-mouse monoclonal antibodies against. SHMT2 (1:300; Abcam, Cambridge, UK), NDUFA4L2 (1:1,000, Abcam, Cambridge, UK) and GAPDH(1:1000, Abcam, Cambridge, UK) overnight at 4 °C with shaking. Then, the membranes were washed in TBST. After that the membranes were washed and incubated with secondary antibody anti-rabbit IgG (1:2000, Santa Cruz, CA, USA) for 1.5 h at room temperature. Data obtained from the western blot experiments were analysed by Bio-Rad Quantity One 1D Analysis software (Bio-Rad, Hercules, CA, USA).

Wang H., Chong T., Li B.Y., Chen X.S, & Zhen W.B. (2020). Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer. Biological Research, 53, 46.

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