Goat anti mouse conjugated with horseradish peroxidase

To compare the activity of HIF-1α WT proteins between mutants A and B and the control under normoxic conditions (20% O₂), the following five groups were transfected into NP cells using Lipofectamine (Invitrogen, Waltham, MA, USA): (1) Desferrioxamine (DFO) only, (2) pcDNA3 only, (3) pHIF-1α WT, (4) pHIF-1α Mut A, and (5) pHIF-1α Mut B. DFO was used as a positive control, because it inhibits both PHD enzyme and FIH-1 enzyme activities [41 (link)]. The transfected cells were lysed using a cell fractionation kit (Cell Signaling Technology, Danvers, MA, USA). The cells were separated into nuclear and cytoplasmic fractions in Nupage 4–12% Bis-Tris gels (Invitrogen, Waltham, MA, USA) and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and washed with phosphate-buffered saline and 0.2% Tween 20 buffer. The membranes were blotted with mouse anti-HIF-1α (Cell Signaling Technology, Danvers, MA, USA), mouse anti-nuclear lamin A/C, or anti-α-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. The blots were treated with goat anti-mouse conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using an enhanced chemiluminescence system (Uppsala, Sweden).

Transfected cells were lysed in 1x Passive Lysis Buffer (Promega, Madison, WI) with protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). SDS dye (6x solution) was added to the lysates and samples were boiled for 10 min. Twenty micrograms of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with a rabbit polyclonal anti-HBZ antiserum (1∶1000), a mouse anti-Flag M2 antibody (1∶5000) (Sigma Aldrich, St. Louis, MO), or mouse anti-Actin (1∶10000) according to standard procedures. Secondary antibodies used included goat anti-rabbit and goat anti-mouse conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1∶2000. Blots were developed using Immunocruz luminol reagent (Santa Cruz Biotechnology) and imaged using the Fuji LAS 4000 imaging system (GE Healthcare Life Sciences, Piscataway, NJ). Densitometry was measured using Multi Gauge version 3.0 software (Fujifilm, Tokyo, Japan).