Fig. 4

a Alignment of the FLCN WT and FLCN mutant sequence on the protein level. The altered amino acid sequence caused by the mutation is shown in red. The alignment was peformed using ClustalOmega [36]. b Equal amounts of FLAG tagged FLCN WT or Mut plasmids were transfected into HEK293T cells and the expression level of both constructs was analysed using Western Blot. The mutant protein (Mut) is less expressed in comparison to the WT protein. Tubulin served as loading control. The results of three independent experiments were analysed by densitometry (n =3, error bars indicate SEM, * = p < 0.05, two tailed t-test).c The FLCN antibody (CellSignaling, #3697) detects the FLCN mutant (delta exon 11). HEK293T cells were transfected with GFP tagged FLCN wildtype (WT), the patient mutation (Mut) or GFP (control). Western blot analyses show that the FLCN antibody detects both the FLCN WT and Mut protein as well as endogenous FLCN (panels on the left side). Expression of all plasmids was verified using an anti-GFP antibody. Tubulin served as loading control