Anti α actinin

Platelet samples for Western blot analysis were prepared by adding 3× Lämmli buffer (200 mM tris/HCl, 15% (v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromphenol blue, 1:10 β-mercaptoethanol) directly in the aggregation cuvettes to stop platelet responses, then boiled at 95 °C for 10 min under gentle shaking. Platelet proteins were separated by electrophoresis using 8% SDS-polyacrylamide gels followed by immunoblotting as previously described [28 (link)].
Phospho-antibodies against Syk S297, Syk Y525/526, Syk Y352, LAT Y191, PLCγ2 Y759, and MARCKS S159/163 (Cell Signaling Technologies, Danvers, MA, USA) were used diluted 1:1000 in 5% BSA or 1:700 for MARCKS. Blots probed with phospho-antibodies were stripped and reprobed with a corresponding antibody detecting the total protein, anti-Syk, or anti-PLCγ2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or other loading controls anti-β-actin or anti-α-actinin (Cell Signaling Technologies, Danvers, MA, USA) diluted 1:1000 in 5% BSA. After incubation with the appropriate secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (BioRad Laboratories, Hercules, CA, USA) enhanced chemiluminescence (ECL) detection was performed using Fusion FX7 (Vilber Loumat GmbH, Eberhardzell, Germany).

Cells were rinsed thrice in PBS before being fixed in 4% paraformaldehyde for 15 minutes, washed in PBS, and incubated for 20 min at ambient temperature in 0.5% Triton X‐100 before being rinsed in PBS. Dropwise injections of bovine serum albumin (5%) were used to the petri dishes, followed by incubation for 30 min at ambient temperature. Next, the cells were incubated in anti‐α‐actinin at 4°C overnight (#3134, Cell Signalling, Danvers, MA, USA), washed, incubated for 30 min at 37°C in the buffer of FITC‐conjugated goat anti‐rabbit IgG away from the light, rinsed with PBS, stained in 6‐diamidino‐2‐phenylindole (DAPI) and examined under (Olympus, Tokyo, Japan).

Anti-phospho-cofilin (Ser-3), anti-cofilin, anti-phospho-MLC (Thr-18/Ser-19), anti-MLC, anti-phopsho-LIMK1 (Thr-508)/LIMK2 (Thr-505), anti-LIMK1, anti-phospho-Src family (Tyr-416), anti-phospho-Src (Tyr-527), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti-α-actinin, anti-paxillin, anti-talin-1, and anti-tensin-2 antibodies were purchased from Cell Signaling Technology. Anti-PCTK3 and anti-vinculin antibodies were from Santa Cruz Biotechnology. Anti-FLAG (M2) antibody was form Sigma-Aldrich. Anti-Strep antibody was from Qiagen. Anti-Halo antibody was from Promega. Anti-GAPDH antibody was from Wako Pure Chemical Industries. Anti-RhoA and Rac1 antibodies and Alexa-555 conjugated-phalloidin were from Cytoskeleton. Anti-Myc antibody was from Enzo Life Sciences.

The NRCMs were fixed in 4% paraformaldehyde for 15 min at room temperature and then permeabilized with PBS containing 0.3% Triton X-100 for 20 min. Next, samples were blocked with 10% goat serum (GTX27481; GeneTex, Sanantonio, TX, USA) for 30 min and then incubated overnight at 4°C with the indicated primary antibodies. The next day, NRCMs were incubated with Alexa-Fluor-conjugated secondary antibodies (ZSGB-BIO,1 : 200) for 2 hours at room temperature. Anti-α-actinin (Cell Signaling, 69758S, 1 : 200) was used to measure the size of NRCMs. Cell colocalization analysis was performed using anti-Keap1 and anti-p62 coincubation and observed under the confocal microscope (TCS-SP8, Leica). DNA was stained with DAPI (1 μM in PBS).

The integrity of the mitochondrial membrane was tested using the fluorescent dye MitoTracker (Life Technologies). Briefly, live cells growing on 60-μm dishes (ibidi) were incubated in media containing 200 nM MitoTracker Red for 15 min at 37 °C in a 5% CO₂ incubator. The cells were then fixed with 1% paraformaldehyde. The fixed cells were permeabilized with 1% (v/v) NP-40 in PBS before staining with antibodies for internal cell markers. The primary antibodies used in this assay were anti-α-actinin (1:200; Cell Signaling), anti-Troponin T (cTNT, 1:200; Millipore), anti-MYL2 (1:200; Millipore) and anti-MYL7 (1:200; Millipore). Confocal microscopic images were then obtained using a Zeiss Confocal Fluorescence Microscope (LSM 510 META, Version 3.2 SP2, Carl Zeiss).