Western Blot Analysis of Cellular Proteins

The harvested cells were lysed in 1% NP-40, 125 mM Tris, pH 6.8, containing a protease inhibitor cocktail (Roche, Basel, Switzerland). For western blotting, 25–50 µg total protein was loaded into 10–12% SDS-PAGE per lane and analyzed by immunoblotting as described previously [46 (link)]. The primary antibodies included: TfR1 antibody (cat#136,800) from Zymed (San Francisco, CA), SDHB (cat#ab178423), cleaved-caspase3 (cat#ab179517) from Abcam (Cambridge, MA), ACO2 (cat#11134-1-AP), NDUFS1 (cat#12444-1-AP), UQCRFS1 (cat#1843-1-AP), NRF2 (cat#16396-1-AP), HO-1 (cat#66743-1-Ig) from Proteintech Group Inc. (Chicago, IN), anti-FTL, FTH, IRP1, and IRP2 (polyclonal, self-made, raised from rabbits) [47 (link)].

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Zhao H., Zhang M., Zhang J., Sun Z., Zhang W., Dong W., Cheng C., Yao Y, & Li K. (2023). Hinokitiol-iron complex is a ferroptosis inducer to inhibit triple-negative breast tumor growth. Cell & Bioscience, 13, 87.

Publication 2023

protease inhibitor cocktail cleaved-caspase3 NDUFS1 UQCRFS1

Antibodies Caspase3 Cat 1 Cells Irp1 Np 40 Nrf2 Protease inhibitor Protein Rabbits Sdhb Sds page Tfr1 Tris

Corresponding Organization :

Other organizations : Nanjing University, Nanjing Drum Tower Hospital, Nanjing Medical University

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Variable analysis

independent variables

NP-40 concentration
Tris concentration
Protease inhibitor cocktail

dependent variables

Protein expression levels of TfR1, SDHB, cleaved-caspase3, ACO2, NDUFS1, UQCRFS1, NRF2, HO-1, FTL, FTH, IRP1, and IRP2

control variables

Total protein amount loaded per lane (25-50 µg)
SDS-PAGE gel percentage (10-12%)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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