Figure 8.

SIDT2 and LAMP2C function synergistically during RNautophagy. (A-D, upper panels) Neuro2a cells seeded in 24-well culture plates were co-transfected with 5 ng of each vector together with carrier DNA (pCI-neo vector) up to 0.1 μg DNA/well using 0.4 μl of 1 μg/μl PEI/well. After 28 h, cells were harvested with lysis buffer and western blotting was performed using anti-SIDT2, anti-LAMP2C and anti-ACTB/β-actin antibodies. (A, lower panel) Degradation of endogenous RNA was assessed in control and SIDT2, LAMP2C or SIDT2 and LAMP2C-overexpressing Neuro2a cells by pulse-chase analysis. Cells were labeled with [³H]-uridine 4 h post-transfection. Radioactivity was expressed as a percentage of degraded RNA. **, P < 0.01 vs Ctrl; ##, P < 0.01 vs LAMP2C, †, P < 0.05 vs SIDT2 (n = 12). (B, lower panel) Degradation of endogenous RNA was assessed in control and 3RS SIDT2, LAMP2C or 3RS SIDT2 and LAMP2C-overexpressing Neuro2a cells by pulse-chase analysis. Cells were labeled with [³H]-uridine 4 h post-transfection. Radioactivity was expressed as a percentage of degraded RNA. No significant difference was observed (n = 8). (C, lower panel) Degradation of endogenous RNA was assessed in control and SIDT2, 2RS LAMP2C or SIDT2 and 2RS LAMP2C-overexpressing Neuro2a cells by pulse-chase analysis. Cells were labeled with [³H]-uridine 4 h post-transfection. Radioactivity was expressed as a percentage of degraded RNA. **, P < 0.01 vs Ctrl; ##, P < 0.01 vs LAMP2C (n = 8). (D, lower panel) Degradation of endogenous RNA was assessed in control and 3RS SIDT2, 2RS LAMP2C or 3RS SIDT2 and 2RS LAMP2C-overexpressing Neuro2a cells by pulse-chase analysis. Cells were labeled with [³H]-uridine 4 h post-transfection. Radioactivity was expressed as a percentage of degraded RNA. No significant difference was observed (n = 8)