Figure 4.
Maspardin deficiency leads to RAB7 hyperactivation. (A) Co-immunoprecipitation analysis of maspardin and RAB7 in CTRL and, as negative control, SPG21 KO HeLa cells. Precipitation of maspardin using protein A-magnetic beads preincubated with an anti-maspardin antibody, followed by endogenous RAB7 detection by western blotting. Beads alone and beads incubated with non-targeting IgG were included as control conditions. (B) Expression of WT, CA, or DN forms of mCherry-RAB7 or mCherry alone in control cells followed by their precipitation using RFP-trap beads. The presence of endogenous maspardin among the coprecipitated proteins was detected by western blotting. n = 4 (two independent experiments, including two CTRL clones [open circles: CTRL1; open squares: CTRL2]). The graph shows mean fold changes ± SD of maspardin signals normalized to the amount of precipitated bait proteins. Two-tailed unpaired t test. ns, nonsignificant; **P < 0.01; ***P < 0.001. (C) Colocalization analysis between endogenously expressed RAB7 (green) and LAMP1 (red) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graphs show the quantifications of RAB7 granularity (right graph) and RAB7-LAMP1 colocalization (left graph, Manders’ coefficients). n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment (shown in light gray). The averaged values are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. ****P < 0.0001. (D) Colocalization analysis between WT, CA, and DN mCherry-RAB7 proteins and LAMP1 (green) in CTRL and SPG21 KO HeLa cells (48 h after transfection). SPG21 WT-myc-flag (blue) was re-expressed in KO cells as a rescue control. Scale bar = 10 µm. The graph shows RAB7-LAMP1 colocalization (Mean Manders’ coefficients ± SD). n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment, as described in B. Two-tailed unpaired t test. ***P < 0.001; ****P < 0.0001. (E) GST pull-down analysis of mCherry-RAB7 in CTRL and SPG21 KO HeLa cells using GST-RILP (RAB7-binding domain [BD]) as bait. CA and DN RAB7 constructs were used as positive and negative controls, respectively. Western blotting was conducted 24 h after transfection using an anti-tdTomato tag antibody. The graph shows the ratios of bound RAB7 to total RAB7 signal calculated in three independent experiments with two CTRL and two KO clones transfected with WT RAB7 in each experiment (n = 6) and one CTRL clone transfected with CA and DN RAB7 (n = 3). Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001. RFP, red fluorescent protein. Source data are available for this figure: SourceData F4.

Maspardin deficiency leads to RAB7 hyperactivation. (A) Co-immunoprecipitation analysis of maspardin and RAB7 in CTRL and, as negative control, SPG21 KO HeLa cells. Precipitation of maspardin using protein A-magnetic beads preincubated with an anti-maspardin antibody, followed by endogenous RAB7 detection by western blotting. Beads alone and beads incubated with non-targeting IgG were included as control conditions. (B) Expression of WT, CA, or DN forms of mCherry-RAB7 or mCherry alone in control cells followed by their precipitation using RFP-trap beads. The presence of endogenous maspardin among the coprecipitated proteins was detected by western blotting. n = 4 (two independent experiments, including two CTRL clones [open circles: CTRL1; open squares: CTRL2]). The graph shows mean fold changes ± SD of maspardin signals normalized to the amount of precipitated bait proteins. Two-tailed unpaired t test. ns, nonsignificant; **P < 0.01; ***P < 0.001. (C) Colocalization analysis between endogenously expressed RAB7 (green) and LAMP1 (red) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graphs show the quantifications of RAB7 granularity (right graph) and RAB7-LAMP1 colocalization (left graph, Manders’ coefficients). n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment (shown in light gray). The averaged values are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Mean ± SD. Two-tailed unpaired t test. ****P < 0.0001. (D) Colocalization analysis between WT, CA, and DN mCherry-RAB7 proteins and LAMP1 (green) in CTRL and SPG21 KO HeLa cells (48 h after transfection). SPG21 WT-myc-flag (blue) was re-expressed in KO cells as a rescue control. Scale bar = 10 µm. The graph shows RAB7-LAMP1 colocalization (Mean Manders’ coefficients ± SD). n = 6 (three independent experiments, including two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment, as described in B. Two-tailed unpaired t test. ***P < 0.001; ****P < 0.0001. (E) GST pull-down analysis of mCherry-RAB7 in CTRL and SPG21 KO HeLa cells using GST-RILP (RAB7-binding domain [BD]) as bait. CA and DN RAB7 constructs were used as positive and negative controls, respectively. Western blotting was conducted 24 h after transfection using an anti-tdTomato tag antibody. The graph shows the ratios of bound RAB7 to total RAB7 signal calculated in three independent experiments with two CTRL and two KO clones transfected with WT RAB7 in each experiment (n = 6) and one CTRL clone transfected with CA and DN RAB7 (n = 3). Mean ± SD. Two-tailed unpaired t test. ns, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001. RFP, red fluorescent protein. Source data are available for this figure: SourceData F4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal