- Grow human HEK293 cells (Cell Biolabs, RV-101) in DMEM medium(Sigma, D5796) supplemented with FBS (Gemini, A59G00J) and Pen Strep (Gibco, 15140-122, lot 1999384), at 37°C and 5% CO2 in 2mL medium on a 6 well plate to 80% confluency.
- Transfect cells with 2µg per well pMXs-Puro murine Cited2 expression plasmid using PEI (Polysciences, 23966-1) following the manufacturer´s instructions.
- Lyse 1x106 cells in 100µl per well cold RIPA (Sigma, R0278).
- Determine total protein content of the lysates using Pierce BCA Protein Assay Kit (Thermo Fisher, 23225).
- Mix 40 µg of total protein with in 10 µL 4x Laemmli Sample Buffer (Biorad, 1610747) and subsequently separate them on a denaturing 12% PAGE gel (Biorad, 1610156) in a Biorad electrophoresis chamber for 1h at 100V.
- Transfer proteins onto Immobilon-PSQ PVDF Membrane (Sigma, ISEQ00010, lot R8CA8256B) with a Western blotting system for 1h at 110V.
- Block the membrane with blocking buffer (5% non-fat milk in TBST) for 1h at RT.
- Incubation with primary rabbit anti-CITED2 antibody (antibodies-online, ABIN2776451, lot QC11500) diluted 1:200 in blocking buffer ON at 4°C.
- Wash membrane 3x for 15min with TBST.
- Incubation with Goat anti-Rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, HRP (Invitrogen, A27036, lot 20310171) diluted 1:2000 in blocking buffer for 1h at RT.
- Wash membrane 5x for 15min with TBST.
- Reveal protein bands using SuperSignal West Dura (Thermo Scientific, 34075, TG268239) on a UVP ChemStudio touch (Analytik Jena, 84997092802).