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Materials and Methods :

Supplementary Material 3 : Qualities of the antisera directed against Hsmar1, SET, THAP4 or Charlie for antibody staining on HeLa cells transiently expressing or not Hsmar1, SETMAR, THAP4 or CHARLIE8. .

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Materials and Methods :

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  1. Supplementary Material 3 : Qualities of the antisera directed against Hsmar1, SET, THAP4 or Charlie for antibody staining on HeLa cells transiently expressing or not Hsmar1, SETMAR, THAP4 or CHARLIE8. Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteinsAhmed Arnaoty, ValérieGouilleux, SophieCasteret, Bruno Pitard,Yves Bigot and Thierry Lecomte Materials and Methods: Cells were plated at a density of 5x104 cells per well in 1 cm2 Lab-TekTM chamber slides (Fisher Scientific, Waltham, MA, USA) and grown in DMEM (Gibco/Life Technologies, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Pasching, Austria) at 37°C in a humidified atmosphere containing 5% CO2 for 24 h to 80% confluence. Transfections were performed as described in the main manuscript using 500 ng of pVAX plasmid and jetPEI™ (Polyplus Transfection, Illkirch, France) at an N/P ratio of 5 in Opti-MEM (Invitrogen/Life Technologies) following the Manufacturer’s instructions. The day after transfection, cells were washed twice with 1X-PBS, fixed with 3.7% paraformaldehyde for 5 min at room temperature (RT), washed three times for 5 min with 1X-PBS at RT, before to be permeabilized in 1X-PBS, 0.2% Triton X100 for 5 min at RT. After 2 washes with 1X-PBS, cells were blocked with 1X-PBS, % bovine serum albumin (BSA) for 30 min at RT. Cells were incubated for 1 h at room temperature with a 1/20, 1/50 or 1/100 of a mouse antiserum (directed against Hsmar1, SET, Charlie 8 and THAP4) in 1X-PBS, and 1% BSA. Cells were then washed three times for 5 min with 1X-PBS before being incubated for 1 h at RT with the secondary antibody (AcII; Alexa Fluor® 488 Goat Anti-Mouse, Invitrogen/Life Technologies). Slides were washed three times for 5 min with 1X-PBS. Nuclei were stained with Hoechst 33342 for 5 min at RT. Slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame CA, USA) for epifluorescencemicroscopicobservation. Slides were analyzed using a Nikon Eclipse Ti and NIS-Elements software. The specificity of the antisera directed against Hsmar1 or SET was monitored using HeLa cells transiently expressing Hsmar1 or SETMAR (SET fused to Hsmar1). Negatives controls were also done for each antiserum to verify the preimmune serum and the quality of the secondary antibody on each type of HeLa (transfected with a plasmid or not).

  2. S3.1 Immunofluorescence results Murine Balbc antiserum N°1 directedagainstHsmar1 on HeLatransfected by pVAX-Hsmar1 plasmid Negativecontrols HelawithoutHsmar1 HelawithHsmar1 - - Antiserum Ac II + + Preimmuneserum (PI) Antisera N°1 HelawithoutHsmar1 HelawithHsmar1 HelawithoutHsmar1 HelawithHsmar1 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  3. S3.2 Immunofluorescence results Murine Balb c antiserum N°1 directedagainstHsmar1 on HeLatransfectedwith the pcDNA-3.1-TOPO-Metnase plasmid Negativecontrols HeLawithoutSETMAR HeLawithSETMAR Antiserum - - Ac II + + Preimmuneserum (PI) Immune serumN°1 HeLawithSETMAR HeLawithoutSETMAR HeLawithSETMAR HeLawithoutSETMAR 1/20 1/20 PI + + 1/50 Ac II + + 1/100 Antiserum + + Ac II + +

  4. S3.3 Immunofluorescence results Murine Balb c antiserum N°2 directedagainstHsmar1 on HeLatransfected by pVAX-Hsmar1 plasmid Negativecontrols HelawithoutHsmar1 HelawithHsmar1 - - Antiserum Ac II + + Preimmuneserum (PI) Antisera N°2 HelawithoutHsmar1 HelawithHsmar1 HelawithoutHsmar1 HelawithHsmar1 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  5. S3.4 Immunofluorescence results Murine Balb c antiserum N°2 directedagainstHsmar1 on HeLatransfectedwith the pcDNA-3.1-TOPO-Metnase plasmid Negativecontrols HeLawithoutSETMAR HeLawithSETMAR Antiserum - - Ac II + + Preimmuneserum (PI) Antisera N°2 HeLawithSETMAR HeLawithoutSETMAR HeLawithSETMAR HeLawithoutSETMAR 1/20 1/20 PI + + 1/50 Ac II + + 1/100 Antiserum + + Ac II + +

  6. S3.5 Immunofluorescence results Murine Swissantiserum N°1 directedagainst SETon HeLatransfected by pVAX-Hsmar1 plasmid Negativecontrols HelawithoutHsmar1 HelawithHsmar1 Antiserum - - Ac II + + Preimmuneserum (PI Antisera N°1 HelawithoutHsmar1 HelawithHsmar1 HelawithoutHsmar1 HelawithHsmar1 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  7. S3.6 Immunofluorescence results Murine Swissantiserum N°1 directedagainst SET on HeLatransfectedwith the pcDNA-3.1-TOPO-Metnase plasmid Negativecontrols HeLawithoutSETMAR HeLawithSETMAR Antiserum - - Ac II + + Preimmuneserum (PI) Antisera N°1 HeLawithSETMAR HeLawithoutSETMAR HeLawithSETMAR HeLawithoutSETMAR 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  8. S3.7 Immunofluorescence results Murine Swissantiserum N°2 directedagainst SETon HeLatransfected by pVAX-Hsmar1 plasmid Negativecontrols Hela-HsMAR1/- Hela-HsMAR1/+ Antiserum - - Ac II + + Preimmuneserum (PI) Antisera N°2 HeLawithoutHsmar1 HeLawithHsmar1 HeLawithoutHsmar1 HeLa-Hsmar1 1/20 1/20 PI + + AcII + + 1/50 1/100 Antiserum + + Ac II + +

  9. S3.8 Immunofluorescence results Murine Swissantiserum N°2 directedagainst SET on HeLatransfectedwith the pcDNA-3.1-TOPO-Metnase plasmid Negative controls HeLawithoutSETMAR HeLawithSETMAR Antiserum - - Ac II + + Preimmuneserum (PI) Immune serumN°2 HeLawithSETMAR HeLawithoutSETMAR HeLawithSETMAR HeLawithoutSETMAR 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  10. S3.9 Immunofluorescence results Murine Balb c antiserum N°2 directedagainst Charlie8on HeLatransfected by pVAX-Charlie8 plasmid Negativecontrols HeLawithout Charlie 8 HeLawith Charlie 8 - - Antiserum Ac II + + Preimmuneserum (PI) Antisera N°2 HeLawithout Charlie 8 HeLawith Charlie 8 HeLawithout Charlie 8 HeLawith Charlie 8 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  11. S3.10 Immunofluorescence results Murine Balb c antiserum N°2 directedagainst Charlie8on HeLatransfected by pVAX-Charlie8 plasmid Negativecontrols HeLawithout Charlie 8 HeLawith Charlie 8 Antiserum - - Ac II + + Preimmuneserum (PI) Antisera N°5 HeLawithout Charlie 8 HeLawith Charlie 8 HeLawithout Charlie 8 HeLawith Charlie 8 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  12. S3.11 Immunofluorescence results Murine Balb c antiserumN°2 directedagainstTHAP4 on HeLatransfected by pVAX-THAP4 plasmid Negativecontrols HeLawithout THAP4 HeLawithTHAP4 - - Antiserum Ac II + + Preimmuneserum (PI) Antisera N°2 HeLawithout THAP4 HeLawithTHAP4 HeLawithout THAP4 HeLawithTHAP4 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

  13. S3.12 Immunofluorescence results Murine Balb c antiserumN°4 directedagainst THAP4 on HeLatransfected by pVAX-THAP4 plasmid Negativecontrols HeLawithout THAP4 HeLawithTHAP4 Antiserum - - Ac II + + Preimmuneserum (PI) Antisera N°4 HeLawithout THAP4 HeLawithTHAP4 HeLawithout THAP4 HeLawithTHAP4 1/20 1/20 PI + + Ac II + + 1/50 1/100 Antiserum + + Ac II + +

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