Fig. 2

Mutations in SGTA NNP region enhance the steady state of a model MLP and promote its accumulation in cytoplasmic inclusions. a Schematic representation of SGTA domain organization alongside a sequence alignment (purple indicates high conservation; expanded version of the alignment is shown in Additional file 1: Figure S1). b Summary of all SGTA-V5 variants designed for this part of the work. c HeLa cells stably expressing OP91 under an inducible promoter were transiently transfected with plasmids encoding either a control (PEX19-V5) or V5-tagged SGTA variants as indicated, and OP91 expression induced. The resulting levels of OP91 and exogenous PEX19/SGTA variants were visualized via Western blotting and fluorescence based detection (LI-COR). d Quantified signals for PEX19-V5 or SGTA-V5 species were normalized to endogenous tubulin, which acted as a loading control and the OP91 levels then expressed relative to the respective normalized V5 signal, with the ratio for OP91/PEX19-V5 set as one. Values show means ± s.e.m. from three independent technical repeats; P was determined by a Student’s t test. e HeLa cells stably expressing OP91 were co-transfected as in (c), the cells fixed, and then labeled for immunofluorescence microscopy using antibodies that recognize either OP91 or the V5 tag on PEX19 and the SGTA variants. Scale bar is 10 μm (see also Additional file 2: Figure S2). f The percentage of cells displaying opsin-positive inclusions were counted from the experiments described in (e) (n ≥ 100 cells/condition from total of three experiments). Error bars show s.e.m., and P is determined by a Student’s t test