G actin f actin in vivo assay kit

F-actin/G-actin ratio in mice was determined with G-actin/F-actin In Vivo Assay Kit (#BK037, Cytoskeleton, Inc, United States) according to the manufacturer’s instructions. Briefly, mouse brain tissue was homogenized in the lysis buffer and incubated at 37°C for 10 min. The lysates were centrifuged at 350 × g for 5 min, and the supernatants were centrifuged at 100,000 × g at 37°C for 1 h. The precipitate is F-actin, and the supernatant is G-actin. Transfer the supernatant containing G-actin to a new tube. Add depolymerization buffer to the precipitate containing F-actin and incubate on ice for 1 h. The extracts containing G-actin and F-actin were assayed by Western blot respectively.

EGF (Cat. No: E9644), phalloidin-TRITC (Cat. No: P1951), actin (Cat. No: A3653), adenosine 5′-triphosphate desmodium (ATP, Cat. No: A1852), and dithiothreitol (DTT, Cat. No: D9779) were purchased from Sigma Chemical Co. (St. Louis, MO). PD153035 (PD) was purchased from Calbiochem (EMD Biosciences Inc., Darmstadt, Germany; Cat. No: 234490). Fluo-3/AM (Cat. No: F1242) and Fluo-4/AM (Cat. No: F14201) were from Molecular Probes (Eugene, OR), the G-actin/F-actinIn Vivo Assay Kit was from Cytoskeleton (Cat. No: BK037), andthe SDS-PAGE kit (Cat.No: P0012A), sample buffer (Cat.No: P0015), actin antibody (Cat.No: AA128) and GAPDH antibody (Cat.No: AG019) were from Beyotime. The mouse monoclonal antibody to fascin (Cat.No: ab78487), anti-myosin light chain antibody [MLC] (Cat.No: ab97891), and anti-Arp3 antibody (Cat.No: ab56817) were from Abcam, and the vinculin antibody (Cat.No: 4650) was from Cell Signaling Technology. The secondary antibodies IRDye 800CW goat anti-mouse IgG (H+L) (Cat.No: 926-32210) and IRDye® 680RD goat anti-rabbit IgG (H+L) (Cat.No: 926-68071) were purchased from LI-COR, and anti-rabbit IgG Fab2 Alexa Fluor(R) 488 Molecular Probes (Cat.No: 4412S) was from Cell Signaling. Fluo-3/AM was purchased from Molecular Probes (Eugene, OR) (Cat. No: F1242).

The G-actin/F-actin In vivo Assay Kit (Cytoskeleton, Denver, CO, USA, BK037) was used to quantify the F- and G-actin pools. Cells were harvested by scraping in lysis buffer. Cell lysates were then incubated for 10 min at 37 °C and centrifuged at 350 g at room temperature for 5 min to pellet unbroken cells. Supernatants were then centrifuged at 90,000 g for 90 min at room temperature to pellet F-actin and leave G-actin in the supernatant. Supernatants were then removed and kept on the side in 5 × SDS sample buffer, while pellets were incubated in F-actin depolymerization buffer on ice for 1 h to allow actin depolymerization to occur before the addition of 5 × SDS sample buffer. G- and F-actin fractions were then analysed by immunoblotting analysis and quantified using the Image Studio Lite program, as the ratio between F- and G-actin levels for each experimental condition.

The actin fractionation was performed according to the ‘G-actin/F-actin in vivo Assay Kit' (Versions 1.1 and 3.5) from Cytoskeleton Inc. (Denver, USA).

The ratio of filamentous (F-) to globular (G-) actin was determined using the G-actin/F-actin in vivo Assay Kit (Cytoskeleton, BK037). Briefly, myoblasts were harvested and lysed 10min at 37°C in Lysis and F-actin Stabilization Buffer. Lysates were cleared by centrifugation at 500 g for 5 min. Subsequently, supernatants were centrifuged at 100,000 g for 1 h at 37°C, which resulted in F-actin in the pellet and G-actin in the supernatant. The F-actin containing pellet was resuspended and solubilized in F-actin depolymerization buffer at a volume equal to the G-actin-containing supernatant volume. Equivalent volumes of supernatant and pellet were resolved by SDS-PAGE and subjected to immunoblot analysis using an anti-pan actin antibody (Cytoskeleton BK037). The F-/G-actin ratio was quantified by using FusionCapt Advance software (Vilber Lourmat).

The fraction of F-to-G actin was assayed using the G-actin/F-actin In Vivo Assay Kit (Cytoskeleton).