Cofilin 1

Non-targeting (cat no. 4390844), ADF (cat no. 4392422; s21737), cofilin-1 (cat no. 4392420; s2936), LIMK1 (cat no. 4390826; s8188), and MYPT1 (cat no. 4390824; s9235) siRNAs were purchased from Thermo Fisher. All siRNA transfections were performed using RNAiMAX (5 µL; Thermo Fisher) and OptiMEM (400 µL; Thermo Fisher). 250,000 cells were collected and seeded in 6-well plates in complete media. siRNAs in RNAiMAX/OptiMEM were added to cells in complete media (2 mL) at a final concentration of 50 nM. Cells were incubated with siRNAs for 3 days.

Non-targeting (cat no. 4390844), cofilin-1 (cat no. 4392420; s2936), and ADF (cat no. 4392422; s21737) locked nucleic acids (LNAs) were purchased from Thermo Fisher. All LNA transfections were performed using RNAiMAX (5 µL; Thermo Fisher) and OptiMEM (400 µL; Thermo Fisher); 100,000 cells were trypsinized and seeded in six-well plates in complete media. After cells adhered (~1 hr), LNAs in RNAiMAX/OptiMEM were added to cells in complete media (2 mL) at a final concentration of 50 nM. Cells were incubated with LNAs for 2 days.

After washing with HBS, cells in six-well glass bottom plates (Cellvis) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in HBS for 20 min at room temperature. Blocking, permeabilization, antibody incubation, and washing were done in HBS with 1% BSA, 1% fish gelatin, 0.1% Triton X-100, and 5 mM EDTA. A 1:250 dilution of ADF (cat no. MA5-25485; Thermo Fisher), cofilin-1 (cat no. MA5-17275; Thermo Fisher), pRLC (cat no. PA5-17727 or MA5-15163; Thermo Fisher), or RLC (cat no. PA5-17624; Thermo Fisher) antibody was incubated with cells overnight at 4°C. After extensive washing, a 1:400 dilution of Alexa Fluor 488-conjugated anti-rabbit secondary antibody (cat no. A-21206; Thermo Fisher) was then incubated with cells for 2 hr at room temperature. Cells were then incubated with a 1:250 dilution of Alexa Fluor 568-conjugated phalloidin (cat no. A12380; Thermo Fisher) and a 1:1000 dilution of DAPI (cat no. D1306; Thermo Fisher). Cells were again extensively washed and then imaged in HBS. Fluorescence intensity of cofilin-1 or ADF was measured in adhered cells with either EGFP-cofilin-1, RFP-cofilin-1, RFP-ADF, or an empty vector fluorescent protein. A minimum of 26 cells over three independent experiments were averaged and normalized to the control in each experimental n-value.