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. Author manuscript; available in PMC: 2020 Mar 1.
Published in final edited form as: Brain Res. 2018 Nov 5;1706:135–146. doi: 10.1016/j.brainres.2018.11.005

Figure 5: SMN depletion alters trafficking of alpha-COP cargoes.

Figure 5:

5a) Increased levels of myc-KA2 at the plasma membrane following knockdown of both alpha-COP (NSC line 4-1-15) and SMN (NSC line 4-56). Plasma membrane levels of EGFR are unchanged after SMN knockdown. 5b) Quantification of myc-KA2 levels at the plasma membrane from three separate transfections. 5c) Total, plasma membrane and endocytosed myc-KA2 in NSC-34 4-56 cells. Again, there is an increase in the level at the plasma membrane after SMN knockdown with doxycycline. To verify even loading, blots were probed with streptavidin-HRP to visualize all biotinylated proteins. 5d) Although there is a decrease in the amount of myc-KA2 that is endocytosed, quantification of three experiments shows that the decrease was not statistically significant by Student’s t-test. 5e) KA2 trafficking is unaffected in NSC-34 cells expressing wildtype or ALS-associated SOD1 (G93A). 5f) Quantification of Lrgm2 transcript levels in knockdown NSC-34 cultures (SMN and alpha-COP respectively) compared to controls. Lrgm2 transcript levels are not significantly affected by loss of SMN or alpha-COP. Quantification of Lrgm2 transcript levels after formaldehyde cross-linked RNA immunoprecipitation (FL-RIP) show decreased Lrgm2 transcript pulled down with alpha-COP when SMN is depleted. As a positive control, we show that decreased amounts of Lrgm2 precipitate after knockdown of alpha-COP.