Protein standard

Aortic proteins were extracted as previously described [29 (link)]. Briefly, the protein from the aortic tissue and cells was extracted with RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA). The protein concentration of aortic proteins was standardized with a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts (25-35 μg) of aortic extracts from WT and Fbn1^mgR/mgR mice without or with DAPT treatment were loaded into 4–20% Criterion TGX precast gels (Bio-Rad Laboratories, Inc.). Following electrophoresis, the gel was transferred onto a 0.45 μm PVDF membrane (Bio-Rad Laboratories, Inc.). The membrane was incubated overnight at 4°C with antibodies directed against Notch3, α-actin, and β-tubulin (1 : 1,000) (Cell Signaling, Beverly, MA). Bound primary antibodies were detected with HRP-conjugated, species-specific, secondary antibodies (Cell Signaling) using the Clarity Western ECL system (Bio-Rad Laboratories, Inc.). The quantification was done using NIH ImageJ software and standardized by internal loading controls. The molecular sizes were determined using protein standards from Fermentas (Glen Burnie, MA).

Aortic proteins were extracted as previously described [22 (link)]. The protein concentration of aortic proteins and SMC cell extracts was standardized with a Bio-Rad protein assay. Equal amounts (25 μg) of aortic tissue extracts or aortic SMC extract from WT and mgR mice were loaded under reducing conditions onto a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences) The membranes were then incubated with the following primary antibodies: TGF-β (Cell Signaling 3711S), KLF4 (Cell Signaling 4038S), α-actin (Santa Cruz Biotechnology, (1A4) sc-32251), Tropoelastin (Elastin Products Co. PR385), MYH11 (Santa Cruz Biotechnology, (H-44) sc-98705, Calponin (Santa Cruz Biotechnology, (FL-297) sc-28545), SM22α (Santa Cruz Biotechnology (H-75) sc-50446), β –actin (Cell Signaling 4967L) and GAPDH (Cell Signaling 5174S). The bound primary antibody was detected with HRP-linked anti-mouse or anti-rabbit IgG (Cell Signaling 7076S and 7074S). Immunoreactive bands were visualized by autoradiography using ECL (Amersham Biosciences). Gelatin zymography for aortic tissue extract and SMC conditioned media was performed as described previously by Longo et al. [23 (link)], with 0.8% gelatin in a 10% SDS-polyacrylamide gel. The molecular sizes were determined using protein standards (Fermentas).

Protein electrophoresis was performed in 12.5% PAG in the presence of SDS via the Laemmli method [44 (link)]. The preparations were aligned by volume (12 µL) in the case of the protein fractions and culture liquids, or by optical density in the case of the cells (10 OD units/mL). The samples were heated in sample buffer at 99 °C for 10 min. A mixture of protein standards (Thermo Fisher Scientific, Waltham, MA, USA) was used as markers: β-galactosidase, 116.0 kDa; BSA, 66.2 kDa; ovalbumin, 45.0 kDa; lactate dehydrogenase, 35.0 kDa; REase Bsp981, 25.0 kDa; β-lactoglobulin, 18.4 kDa; and lysozyme, 14.4 kDa. Electrophoresis in a concentrating gel was performed at 90 V, and in a separating gel, at 180 V. Protein bands in the gel were detected using imidazole staining and ZnCl₂ solutions [45 (link)], or Coomassie Brilliant Blue R-250 (Serva, Heidelberg, Germany).

To evaluate the levels of active MMPs in the supernatant of the HCLE treated with tears extract, gelatin Zymography was performed using 8% acrylamide gel with a final concentration of 1 mg/mL gelatin. An equal concentration of cell soup was mixed with 4x loading eye (no reducing agent and no heating) and incubated for 5 min. An equal amount of protein and pre-stained protein standards (Thermo Fischer Scientific, Waltham, MA, USA, Cat no. 26616) was loaded in each well of the gel and separated via electrophoresis. Next, the gel was incubated in 1% triton-X 100 for 30 min twice, followed by incubation in developing buffer (composition) overnight at 37 °C. The next day, developing buffer was removed and the gel was stained with Coomassie blue stain for 10 min. The gel was washed and incubated in de-staining buffer to remove the excess stain. The clear band in the gel indicates the MMPs activity. The bands were quantified using ImageJ software and were plotted as a bar graph.