Additional file 2 of Nexilin promotes calcium-dependent endo-lysosomal fission required for retrograde transport

Supplementary Material 2: Figure S2. Nexilin regulates the size of Rab7b-positive LE/Lys in different cell lines. A U2OS, PC3 and HeLa cells were lysed and subjected to Western blot analysis using antibodies against nexilin, and against tubulin as loading control. B PC3 cells were transfected with siRNAs against nexilin or non-targeting control siRNA, and transiently transfected for GFP-Rab7b before live cell imaging. Scale bar: 10 μm. C Quantification of the number of large LE/Lys with an area over 3 μm per cell after nexilin knock down. Data represents the mean ± s.d. for 2 independent experiments (n = 30 cells in total per condition). D HeLa cells were transfected with siRNAs against nexilin or non-targeting control siRNA and transiently transfected with GFP-Rab7b before live cell imaging. Scale bar: 10 μm. E HeLa cells transfected with siRNA against nexilin or non-targeting siRNA were lysed and subjected to Western blot analysis using antibodies against nexilin and tubulin. F Amino acid sequences of nexilin. The phosphorylation site (S) and the corresponding mutated amino acids are indicated in red. G Quantification of colocalisation of Rab7b with nexilin from U2OS cells co-transfected with mCherry-Rab7b and GFP-Nexilin wild type (wt), S442E (phosphorylation-mimic mutant) or S442A (phosphorylation-null mutant), and imaged using a super resolution spinning disk microscope. Data represents the mean ± s.d. from 3 independent experiments (n ≥ 19 cells in total per condition), one-way ANOVA followed by Tukey’s post-hoc test was applied for statistical analysis