Ab81046

Total protein was extracted from the indicated cells or tissues using RIPA lysis buffer (Beyotime, China), and the protein concentration was measured using a BCA protein assay kit (Beyotime, China). The protein was separated by 10% SDS-PAGE and subjected to western blotting, as described previously [43 (link)]. The primary antibodies used in western blotting were as follows: LIMK1(1:1000, ab81046, Abcam), p-cofilin (1:1000, #3313, CST), cofilin (1:1000, #5175, CST), p-CREB(Ser133) (1:1000, #9198, CST), CREB (1:1000, #9197, CST), MMP2(1:1000, ab97779, Abcam), ITGB1(1:1000, ab134179, Abcam) and COL1A1(1:1000, #72026, CST); GAPDH (1:1000, Boster, Wuhan, China) was used as a loading control. Horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG antibodies (1:3000, Boster, Wuhan, China) were used as secondary antibodies, and the protein expression levels were visualized using an enhanced chemiluminescence system (Bio-Rad, Hercules, EDAUSA). Band intensities from three biological experiments were quantified by densitometry using ImageJ software.

Isolated left hippocampus tissue was lysed in 100 µL radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, Jiangsu, China) plus protease inhibitors. Total protein (50 µg) was loaded into 10% SDS-PAGE gels, electrophoresed, and then transblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA) in a Tris-glycine transfer buffer. After being blocked in 5% milk in PBST for 1 h at room temperature with shaking, membranes were incubated with antibodies overnight at 4°C against the following: anti-LIMK1 antibody (dilution, 1 : 1000; ab81046, Abcam) and anti-LIM kinase 1 antibody (phospho-Thr508) (dilution, 1 : 1000; ab131341, Abcam) and β-actin (dilution, 1 : 1000; ab189073; Abcam). The following day, membranes were incubated in 5% milk (in TBST) with an anti-goat or anti-mouse IgG antibody (dilution, 1 : 5000; PerkinElmer Life Sciences, Waltham, MA) for 1 h at room temperature with shaking. Membranes were washed a minimum of four times (10 min per wash) in PBST between each antibody treatment. Detected bands were visualized using enhanced chemiluminescence and images were captured using a Bio-Image system (Bio-Rad Laboratories, Inc., Hercules, USA). Western blotting was repeated three times.

Protein samples were collected from cell lysates. Protein was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and then were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). At room temperature, these membranes were blocked in PBS containing 5% nonfat milk for 1 h, and then incubated with primary antibodies overnight at 4°C. The following were the primary antibodies in this research: anti-MEX3A (dilution: 1:500, Abcam, UK, ab79046), anti-RhoA (dilution: 1: 500, Abcam, UK, ab187027), anti-ROCK1 (dilution: 1:1000, Abcam, UK, ab134181), anti-LIMK1 (dilution: 1:500, Abcam, UK, ab81046), anti-β-actin (dilution: 1:1000, Abcam, UK, ab8226), anti-RhoA (phospho S188) (dilution: 1:1000, Abcam, UK, ab41435), anti-phospho-ROCK1 (Tyr913) (dilution: 1:1000, Invitrogen, USA, PA5-105054) and anti-phospho-LIMK1 (Thr508) (dilution: 1:1000, Invitrogen, USA, PA5-104925). After being washed with TBST, these membranes were incubated with secondary antibody at 25°C for 1 h. Protein blots were visualized using enhanced chemiluminescence (Millipore, USA) [14 ].