Extended Data Figure 4. HERV-K Gag/Capsid antibody validation and staining (supporting Fig. 3).

a) Immunofluorescence analysis of hECCs (NCCIT) and hESCs (H9) stained with DAPI (blue), OCT4 (green), Gag/Capsid (red), or IgG control (bottom). White boxes indicate regions shown in higher magnification/merge (right) Shown are representative fields of three independent experiments.

b) Sensitivity of HERV-K Gag/Capsid antibody immunoblot signal to HERV-K knockdown. hECCs were transfected with one of three independent siRNA pools targeting HERV-K Gag or with a control, non-targeting pool (synthesized against RFP) and total protein was analyzed by immunoblotting with anti-Env and anti-Gag/Capsid antibodies. 1:2 serial dilution of total protein was loaded, as indicated. Blots were stripped and re-probed with TBP as a loading control. Shown is a representative result of two independent experiments.

c) Sensitivity of HERV-K Gag/Capsid antibody immunofluorescence signal to siRNA knockdown of Gag/Capsid (top) or control siRNA targeting RFP (bottom). Shown is a representative result of three fields of view.

d) Immunoflourescence of naïve ELF1 hESC with antibodies against OCT4 (green), HERV-K Gag/Capsid (pink), DAPI in blue. Region marked with white box on left is shown with larger magnification (bottom).

e) Another representative example of immunoflourescence of human blastocyst with DAPI (blue), OCT4 (green), Gag/Capsid (red) shown (n=19 blastocysts), DPF=5–6.