Skip to main content
. 2023 Jun 12;24(8):e56834. doi: 10.15252/embr.202356834

Figure EV4. Data supporting the extreme N terminus of RIF1 binding SHLD3 through polar residues.

Figure EV4

  1. Immunoblot of whole cell extracts of U2OS 2‐6‐3 cells transfected with plasmids encoding the indicated mCherry‐LacR‐fused RIF1 truncations. Lysates were probed for mCherry and tubulin (loading control). IB: immunoblot, EV: empty vector. *: nonspecific band.
  2. Representative micrographs (of three biologically independent experiments) of the LacR/LacO assay using the indicated mCherry‐LacR‐fused RIF1 variants to evaluate their ability to recruit eGFP‐SHLD3 to chromatin shown in Fig 5A.
  3. Immunoblot of whole cell extracts of U2OS 2‐6‐3 cells transfected with plasmids encoding the indicated mCherry‐LacR‐fused RIF1N (residues 1–967) alanine variants. Lysates were probed for mCherry and tubulin (loading control). *: nonspecific band.
  4. Representative micrographs (of three biologically independent experiments) of the LacR/LacO assay using the indicated mCherry‐LacR‐fused RIF1N variants to evaluate their ability to recruit eGFP‐SHLD3 to chromatin shown in Fig 5B. WT: wild‐type.
  5. Immunoblot of whole cell extracts of U2OS 2‐6‐3 cells transfected with plasmids encoding the indicated mCherry‐LacR‐RIF1N and eGFP‐SHLD3C variants. Lysates were probed for mCherry, eGFP, and tubulin (loading control). *: nonspecific band.
  6. Representative micrographs of the LacR/LacO assay using the indicated mCherry‐LacR‐RIF1N and eGFP‐SHLD3C variants to evaluate their colocalization at LacO arrays shown in Fig 5D.
  7. Sanger sequencing chromatograms of PCR products amplified from U2OS 2‐6‐3 cells with and without subjecting it to base editing to introduce endogenous D28N mutations. A single clone was isolated that contained the desired mutation (D28N‐15). Red boxes highlight induced mutations. The first heterozygous G > A mutation is silent. The second homozygous G > A mutation results in the desired D28N substitution.
  8. Immunoblot of whole cell extracts of U2OS 2‐6‐3 cells with or without base editing to introduce endogenous D28N mutation. Lysates were probed for RIF1 and tubulin (loading control).

Source data are available online for this figure.