Fig. 3: N-α-SUMOylation of CFL1 is reversible.

The SUMOylation of CFL1 was inhibited by the knockdown of SAE1 (a), SAE2 (b), and Ubc9 (c), but enhanced by the knockdown of SENP1 (d). HEK-293T cells were transiently transfected as indicated. Cell lysates were used for De-IP by anti-CFL1 antibody followed by IB using anti-SUMO1 and anti-CFL1 antibodies. The original lysates were analyzed by IB using anti-CFL1, anti-SAE1 (a), anti-SAE2 (b), anti-Ubc9 (c), and anti-SENP1 (d) antibodies for input, as well as anti-GAPDH antibody for loading control. e CFL1 is physically associated with SAE1, SAE2, Ubc9 and SENP1. Lysates from HEK-293T cells transiently transfected with the vector control (−), CFL1^WT-HA, CFL1^25KR-HA, HA-DUSP6, and His-SUMO1 as indicated for 24 h were subjected to IP with anti-HA antibody, followed by IB with anti-SAE1, anti-SAE2, anti-Ubc9, anti-SENP1 and anti-HA antibodies. The original lysates were analyzed by IB using anti-HA, anti-SAE1, anti-SAE2, anti-Ubc9 and anti-SENP1 antibodies for input, and anti-GAPDH antibody for loading control. f Similar to (e) but with expression of the vector control (−), CFL1^WT-HA, CFL1^2KR-HA, CFL1^2KQ-HA, and His-SUMO1 as indicated. g–i Quantification of Western blots of (a)–(f). g Summary of SUMOylated CFL1 to total CFL1 presents the mean ± SEM of three independent experiments in (a)–(d); h Summary of coimmunoprecipitated (Co-IPed) SAE1, SAE2, Ubc9 and SENP1 normalized to total CFL1 or DUSP (HA) presents the mean ± SEM of three independent experiments in (e); i Summary of coimmunoprecipitated (Co-IPed) SAE1, SAE2, Ubc9 and SENP1 normalized to total CFL1 (HA) presents the mean ± SEM of three independent experiments in (f); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one-way ANOVA with Tukey’s multiple comparison test. All blots represent ≥3 independent experiments. The exact p value and source data are provided as Source Data file.