Anti sm α actin

Femoral arteries were dissected and embedded in paraffin, and cross-sections were cut at 200 μm intervals. The sections were deparaffinized and treated with citrate buffer for antigen retrieval, followed by incubation in 3% H₂O₂. The sections were then blocked with Dako serum-free blocking solution (Dako, X090930) and incubated with primary antibody at 4°C overnight. Next, the sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. VECTASTAIN ABC HRP kit (Vector Laboratories, PK-4000) and DAB peroxidase substrate kit (Vector Laboratories, SK-4100) were used for detection. Hematoxylin was used for nuclear counterstaining. The primary antibodies used in this study are anti-SIK3 (LSBio, LS-B9603), anti-SM-α-actin (Dako, M0851), anti-PCNA (Santa Cruz, sc-56), and anti-Mac2 (Cedarlane, CL8942AP). The matched IgG was used for negative control. Slides were visualized, and images were captured by Zeiss AxioImager M1 microscope.

Lysates were produced from VSMC cultures and diluted with Laemmli buffer (62.5mM TRIS-HCl pH 6.8, 25% (w/v) Glycerol, 2% (w/v) SDS and 0.01% (w/v) Bromophenol blue). Protein concentrations were determined using a Micro BCA Protein Assay (Pierce Thermoscientific). Equal quantities of protein were loaded into each well of a Mini-PROTEAN TGX gel (BIO-RAD) and SDS-PAGE was performed using the mini-PROTEAN tetra system (BIO-RAD). Sample proteins were then transferred to an Immuno-blot PVDF membrane (BIO-RAD) and detected using protein specific antibodies, horseradish peroxidase-conjugated antibodies and Pierce ECL Western Blotting Substrate (Pierce Thermoscientific).
The following antibodies were utilized: anti Phospho-p44/42 MAPK (ERK1/2) 1:2000 (Cell signaling), anti p44/42 MAPK (ERK1/2) 1:1000 (Cell signaling), anti phospho-STAT-3 1:2000 (Millipore), anti beta Actin as loading control 1:1000 (Abcam), anti SMα-actin 1:1000 (Dako), anti RANKL (R&D Systems), anti Rabbit IgG Peroxidase Conjugate (Sigma), anti goat IgG HRP (Santa Cruz Technologies, Santa Cruz, CA), anti mouse IgG peroxidase (Sigma).