Fig 1: Skeletal muscle biopsies: immunofluorescence studies for RILP and LAMP2 antibodies. (A) In RILP mutated tissues the positive reaction for RILP/CY3 was visible both in the form of granular aggregates of variable extension and irregular profile located near the sarcolemma or scattered in the sarcoplasm (F2_II-4) and in the form of more or less extended subsarcolemmal linear reaction (F1_II-5). Immunofluorescence for LAMP2/FITC followed similar patterns of distribution but to a lesser extent and intensity. (B) The LOPD family members not mutated for RILP-positive RILP/CY3 granules appeared more intensely fluorescent than in mutated tissues with a similar subsarcolemmal and sarcoplasmic pattern of distribution. A non-LOPD vacuolar myopathy (CNT1) and a non-myopathic condition (CNT2) were used as external controls. In CNT1, a faint subsarcolemmal continuous signal was observed in the majority of fibres, while CNT2 showed almost no immunofluorescence. Nuclei were counterstained with DAPI (blue). Scale bar: 200 µm.
Fig 2: DHA increases the basal level of autophagy. Protein levels of ATG5, ATG12, ATG16L1, Beclin1, ATG14, p-ATG14 (Ser29), Rab7, RILP, Lamp1, CTSB, p-mTOR(Ser2448), mammalian target of rapamycin (mTOR), ULK1, p-GSK3ß (Ser9), GSK3ß in extracts of whole brain tissue were detected by WB. Representative Western blots are presented in panels (A,C,E,G), and quantification of the results is shown as the mean values ± SD in panels (B,D,F,H). *p < 0.05, **p < 0.01, and ***p < 0.001 between two groups. One-way analysis of variance/Newman–Keuls test was performed for all WB data.
Fig 3: Immuno-localization of the RILP wt (RILP-284G-GFP) and mutant (RILP-284S-GFP) proteins in the HeLa cells wt and mutant. RILP proteins were fused to the green fluorescent protein (GFP), and LAMP2 and p62 proteins were stained using anti-LAMP2/CY3 and anti-p62/CY3. F-actin fibres were stained with rhodamin phalloidin and nuclei were stained with Hoechst. Slides were visualized with a Nikon Confocal Microscope A1R at 60× magnification.
Fig 4: Autophagosomes in hippocampal axons mature slowly. (A–F) Example kymographs and quantification of LC3 comigrating with Rab7 (A and B), LAMP1 (C and D), or LysoTracker (LysoTr) Deep Red dye (E and F) in different subaxonal regions. n = 8–12 neurons. Two-way ANOVA with Tukey’s multiple comparisons test (distal vs. mid: Rab7, P = 0.9956; LAMP1, P = 0.4166; LysoTracker, P = 0.0709; distal vs. proximal: Rab7, P = 0.9088; LAMP1, P = 0.0264; LysoTracker, P = 0.0280; mid vs. proximal: Rab7, P = 0.8655; LAMP1, P = 0.3205; LysoTracker, P = 0.8623.) (G and H) Example micrographs with line scans and quantification demonstrating RILP association with autophagosomes of differing maturity. n = 8–9 neurons. Two-way ANOVA with Sidak’s multiple comparisons test (mid, P = 0.4894; proximal, P = 0.0403). Dashed yellow lines indicate line scan. Scale bar, 1 µm. Bars throughout show mean ± SEM. *, P < 0.05.
Fig 5: Impaired Rab7 effector interactions in 22L-N2a cells.A, N2a and 22L-N2a cells were transfected with GFP-tagged WT-Rab7 and subjected to immune pull-down with anti-GFP nanobody-coupled agarose beads and subjected to immunoblotting with anti-RILP antibody. The quantitative analysis of the RILP signal normalized to the amount of GFP-tagged WT-Rab7 levels after stripping these membranes with methanol is also shown. B, immunofluorescence images depicting the localization of lysosomes, by probing with anti-Lamp1 antibody in N2a and 22L-N2a cells as visualized by the maximum intensity projections of the z-stack sections, obtained by confocal microscopy. All confocal images are representative of five fields of view from three independent experiments. Statistical significance was evaluated using the unpaired t test. The dashed and dotted lines in the violin plot depict the median and the lower and the upper quartiles of values, respectively. C, N2a and 22L-N2a cells were transfected with GFP-tagged WT-Rab7 and subjected to immune pull-down with anti-GFP nanobody-coupled agarose beads and subjected to immunoblotting, with anti-ubiquitin antibody. Quantitative analysis of the Ub-Rab7 signal normalized to the amount of GFP-tagged WT-Rab7 levels after stripping these membranes with methanol is also shown. Unpaired t test was used to analyze the statistical significance between different groups from three or more independent experiments, and the results are summarized in Table S1. The error bars indicate the standard deviation.
Supplier Page from Abcam for Anti-RILP antibody