Mouse anti calponin antibody

For western blots, HASM cells were lysed for 2 min on ice in M-PER lysis buffer (Thermo Scientific) supplemented with a cocktail of protease inhibitors (Sigma-Aldrich) and centrifuged for 20 min to collect protein lysate. For immunoblotting, 10 μg of lysate from each sample was separated on 6% SDS polyacrylamide gel and electro-transferred onto PVDF membrane (Amersham Pharmacia, ON). The membrane was blocked at room temperature for 2 h with 5% skim milk, incubated with mouse anti-MLCK (K36 clone) polyclonal Ab (Sigma-Aldrich), or mouse anti-calponin antibody (Sigma-Aldrich) at room temperature for 2 h, followed by secondary antibody HRP-goat anti-mouse IgG prepared in 1% skim milk. All the blots were developed by enhanced chemiluminiscence as recommended by the supplier (Amersham Pharmacia, ON). β-actin was used as internal control. The intensity of smMLCK, myosin, calponin and β-actin bands was determined by using AlphaEase FC software version 3.1.2 relative to control loading levels. For signaling, the intensity of phosphorylated forms of ERK, JNK, P38, myosin and smMLCK was normalized with the intensity of total ERK1/2, JNK and P38, respectively.

Collagenase type II, Elastase, and Exisulind were obtained from Sigma-Aldrich co. (St Louis, MO, USA). PDGF-BB was purchased from R&D systems (Minneapolis, MN, USA). Goat anti-PKG I, rabbit anti-VE-cadherin, mouse anti-osteoponin, rabbit anti-CD31, and goat anti-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-VASP (Ser239), rabbit anti-t-Akt, and mouse anti-p-Akt (Ser473) were purchased from Cell Signaling (Berkely, MA, USA). Mouse anti-calponin antibody was obtained from Sigma-Aldrich co. (St Louis, MO, USA). Mouse anti-α-SMA antibody was purchased from Abcam (Cambridge, MA, USA). The secondary antibodies to each primary antibody were as follows. Anti-mouse IgG HRP conjugated and anti-rabbit IgG HRP conjugated antibodies were obtained from Promega (Madison, WI, USA). Anti-goat IgG HRP conjugated antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse IgG Alexa flour 488 and anti-rabbit IgG Alexa flour 594 secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Alzet osmotic pump was purchased from Durect corporation (Cupertino, CA, USA).