Fig. 6.

Role of GON7 on KEOPS complex stability. a Immunoblot analysis of HEK293T cell lysates expressing either 2HA-tagged GON7 or V5-tagged LAGE3 alone or co-expressing both proteins. Anti-HA and anti-V5 antibodies were used to assess GON7 and LAGE3 expression, respectively, with α-tubulin used as loading control. b Representation of cycloheximide chase experiments by fitting a one-phase exponential decay curve to experimental data (one representative experiment is shown in Supplementary Fig. 9) (mean ± s.e.m. of n = 3 experiments). HEK293T cells were transfected with either 2HA-tagged GON7 or V5-tagged LAGE3 alone or with both proteins before being subjected to treatment with 100 µg/ml cycloheximide for the indicated time points in order to assess rates of protein degradation followed by western blotting of the cell lysates for both proteins with anti-HA and anti-V5 antibodies, respectively. GON7 and LAGE3 protein levels were normalized to those of α-tubulin at each time point. c Western blot analysis of protein expression level of the five KEOPS subunits in lymphoblastoid cell lines from two unaffected relatives (A.II-1 and A.II-5), four individuals with the GON7 mutation p.Tyr*7, one individual with the OSGEP mutations p.Arg325Gln and p.Arg280His (individual «N2705» described in Braun et al.¹³), and one individual with GAMOS linked to WDR73 mutations (individual A.II-4 described in Colin et al.¹⁵). One representative western blot is shown (three independent experiments were performed). α-Tubulin was used as a loading control. Source data are provided as a Source Data file