Cofilin 1

Cell lysates of equal protein concentration were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with skimmed milk (5%) and then incubated with primary antibody against specific target proteins of interest. Antibodies against ROCK1, LIMK1, Cofilin-1, and p-Cofilin-1 were purchased from Cell Signaling Technology (CST, Danvers, MA, USA) and were used at a dilution of 1:500. Antibodies against N-cadherin, E-cadherin, Vimentin, and GAPDH were purchased from Abcam (Cambridge, MA, USA) and were all used at a dilution of 1:1000. Subsequently, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, the EasyBlot ECL kit (Sangon, China) was used to visualize the protein bands.

The mouse brain tissue samples were lysed in lysis buffer (50 mM Tris, pH 7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate and 10 mM sodium β-glycerophosphate, supplemented with protease inhibitors cocktail), and centrifuged for 15 min at 16,000 g. The supernatant was boiled in SDS loading buffer. After SDS-PAGE, the samples were transferred to a nitrocellulose membrane. Primary antibodies to the following targets were used: GAPDH (Proteintech, 60004-1-Ig, 1:8000), TH (Sigma-Aldrich, AB152, 1:1000), p-Ser129 α-synuclein (Cell Signaling Technology, 23706 S, 1: 1000), Cofilin 1 (Cell Signaling Technology, 5175 S, 1:1000). The membranes were incubated with primary antibodies overnight at 4 °C. The membranes were washed 3 times in PBST and incubated with HRP-conjugated secondary antibodies. The signals were developed using enhanced chemiluminescent (ECL) substrates. The experiment was repeated at least three times. All blots derived from the same experiment and were processed in parallel.

Total cells were lysed with 2% SDS solution plus protease and phosphatase inhibitors (Thermo Scientific). Protein concentrations were tested by BCA kit and equivalent proteins were loaded into SDS-PAGE. Following western blots were performed according to standard procedures. The primary antibodies were list as follow: MTCH2 (Proteintech, Cat#16888-1-AP), Beta actin (Boster Biological Technology, Cat#BM0627), PDHE1-A (Abcam, Cat#ab110334), GFAP (Millipore, Cat#G3893), Tom20 (Santa Cruz Biotechnology, Cat#sc-136211), Tom40 (Abcam, Cat#ab185543), MMP-9 (Cell Signaling Technology, Cat#3852), N-cadherin (Cell Signaling Technology, Cat#4061), Vimentin (Cell Signaling Technology, Cat#5741), 4-HNE (Abcam, Cat#ab48506), BcL-2 (Santa Cruz Biotechnology, Cat#sc-7382), Bax (Millipore, Cat#06-499), phospho-AKT (Thr308) (Cell Signaling Technology, Cat#2965), phospho-AKT (Ser473) (Cell Signaling Technology, Cat#4060), AKT (Cell Signaling Technology, Cat#9272), phospho-S6 (Ser240/244) (Cell Signaling Technology, Cat#2215), S6 (Cell Signaling Technology, Cat#2217), phospho-4EBP1 (Thr37/46) (Cell Signaling Technology, Cat#9459), total OXPHOs rodent antibody cocktail (Abcam, Cat#110413), phospho-Cofilin1 (Ser3) (Cell Signaling Technology, Cat#3313), Cofilin1 (Cell Signaling Technology, Cat#5175), PARP (Abclonal, Cat#A0942) and cleaved caspase 3 (Cell Signaling Technology, Cat#9661).

Cell treated as indicated were harvested. The protein concentration was analyzed by BCA protein Assay Reagent (Sangon Biotech, Shanghai, China). Soluble lysates containing about 20 μg proteins per sample were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. The membrane signals were detected using an Enhanced Chemiluminescent Western Blotting Detection System (Millipore, Billerica, MA, USA) in accordance with the manufacturer's instruction. Antibodies against ROCK, PTEN, p-PI3K, PI3K, p-Akt, Akt, cofilin-1, p-cofilin-1, and GAPDH were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cleaved ROCK were purchased from Abcam (Cambridge, MA, USA). An anti-PP1 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).