Fig 1: KD of Neurl2 blocks SMN degradation to increase SMN protein levels(A) SMA patient-derived fibroblasts were transfected with siRNA targeting Neurl2. Gene expression and western blot analysis show that Neurl2 KD resulted in increased SMN levels. Data were analyzed using an unpaired t test (Neurl2 mRNA: p < 0.0001; SMN protein: p < 0.0001). n = 5.(B) SMA patient fibroblasts were transfected with GFP-Neurl2 or Neurl2-GFP. SMN was immunoprecipitated, and Neurl2 was associated, confirming their interaction.(C) Patient fibroblasts were transfected with siRNA targeting Neurl2, Mib1, or both. Western blot analysis of SMN levels 48 h following transfection. Data were analyzed using a one-way ANOVA (F(5,24) = 65.53, p < 0.0001) followed by post hoc Tukey’s multiple comparisons test (control versus Neurl2: p = 0.0305; control versus Mib1: p < 0.0001; Mib1 versus Mib1 + siNeurl2: p < 0.0001; Mib1 versus Mib1 + siNeurl2 + Neurl2res: p = 0.7566). n = 5.(D) Patient cells were transiently transfected with 1 μg HA-ubiquitin and FLAG-Mib1, siRNA targeting Neurl2, or siRNA-resistant Neurl2 (Neurl2res). SMN was immunoprecipitated, and SMN ubiquitination was determined by western blot using an anti-polyubiquitin (FK1) antibody. n = 3.(E) Cell-free SMN ubiquitination assay. Recombinant SMN was incubated with E1 and E2 (UBCH5B or UBCH1) enzymes with or without Mib1 or Neurl2 and ubiquitin for 1 h at 37°C. Western blots were probed with an FK1 antibody. Data were analyzed using one-way ANOVA (F(3,16) = 8.874, p = 0.0011) followed by post hoc Tukey’s multiple comparisons test (control versus siNeurl2: p = 0.0060; control versus siMib1: p = 0.0027; control versus siNeurl2 + siMib1: p = 0.0025; siNeurl2 versus siMib1: p = 0.9785; siNeurl2 versus siNeurl2 + siMib1: p = 0.9740; siMib1 versus siNeurl2 + siMib1: p > 0.9999). n = 5.(F) SMN protein stability following siRNA KD of Neurl2, overexpressing FLAG-Mib1, or both. Data were analyzed using one-way ANOVA (8 h: F(2,9) = 9.920, p = 0.0053; 16 h (F(2,9) = 18.93, p = 0.0006) followed by post hoc Tukey’s multiple comparisons test (8 h: control versus siNeurl2: p = 0.0447; 16 h: control versus siNeurl2: p = 0.0036). n = 4.All data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.