Vaspps239

Snap-frozen lung tissues were homogenized (10% w/v) in solution of 0.1 M Na phosphate buffer containing 5 mM EDTA (pH 7.4) using TissueLyser II (Qiagen, Germantown, MD) and centrifuged at 12000 rpm for 20 min to prepare post mitochondrial supernatants. Protein concentrations were measured using Bio-Rad protein reagent (Bio-Rad, Hercules, CA). Equal amounts of post mitochondrial supernatants were dissolved in SDS sample buffer and proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-human polyclonal antibodies targeting profilin1 (catalog# 3237), cofilin1(catalog# 5175), VASP (catalog# 3112), VASP^pS239 (catalog# 3114) (Cell Signaling Technology, Inc., Danvers, MA), VASP^pS157 (catalog# sc-23,506-R) (Santa Cruz, Dallas, Texas) (dilution, 1:500), and rabbit monoclonal antibodies targeting GAPDH (D16H11) (catalog #5174)(1:10,000) (Cell Signaling Technology, Inc., Danvers, MA). Finally, membranes were probed with horseradish peroxidase (HRP)-conjugated secondary antibody (dilution, 1:1000) (BD Pharminogen®, Franklin lakes, NJ) for 1 h at room temperature and specific bands were visualized employing Amersham™ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and the band intensity was measured by densitometry.

Tissues were processed for immunofluorescence (IF) labelling using standard protocols [30 (link)]. Tissue sections were stained with primary anti-human polyclonal antibodies targeting profilin1 (catalog# 3237), cofilin1(catalog# 5175), VASP (catalog# 3112), VASP^pS239 (catalog# 3114) (Cell Signaling Technology, Inc., Danvers, MA), VASP^pS157 (catalog# sc-23,506-R) (Santa Cruz, Dallas, Texas) (dilution, 1:500), followed by secondary antibodies Alexa 488 labeled IgG (1:1000) and nuclei stained with DAPI (Invitrogen, Carlsbad, CA). Images were taken using Carl Zeiss’s Axio Scope A1 Polarized Light Fluorescent Microscope (Carl Zeiss, Jena, Germany) with 40X magnification and identical settings. Four photomicrographs were obtained from each tissue section assessed independently and averaged for each individual thus creating an n = 8 for each diagnosis. Intensity of image color was quantified using NIH image J software.