Hrp linked anti rabbit or anti mouse igg

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).

siRNA-transfected HeLa cells were lysed with Cell Lysis Buffer (Cell Signaling). The lysates were separated by electrophoresis in a 7.5–15% polyacrylamide gel (DRC) and blotted onto a PVDF membrane (Millipore). The membrane was blocked with 3% BSA in TBS, incubated with primary antibody diluted in Can Get Signal Solution I (TOYOBO) overnight, incubated with HRP-linked anti-mouse or anti-rabbit IgG (Cell Signaling) diluted in TBS-T for 1 hour, and developed with ECL (GE Healthcare). Images were visualized using an ImageQuant LAS-4000 imaging system (Fujifilm).

Each protein extract was adjusted to a 2 μg/μL concentration with the addition of Laemmli sample buffer (4X concentrate, Bio-Rad, Hercules, CA, USA), heated for 5 min at 95 °C, and separated on precast polyacrylamide gradient gels (7.5–15%). After transfer to 0.2 μm polyvinylidene difluoride membranes (PVDF, Bio-Rad), the membranes were blocked in either 5% (w/v) nonfat dried milk or 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO, USA) diluted in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T), and probed overnight at 4 °C with the respective primary antibodies, as follows: mouse anti-actin (1:2500), rabbit anti-LDHA (1:1000), rabbit anti-microtubule-associated protein 1B/2B light chains 3B (MAP-LC3) (1:1000) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HIF-1α (1:250), rabbit anti-PDK1 (1:1000), rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:500), and rabbit anti-caspase 3 (1:250) (Abcam, Cambridge, UK). Membranes were subsequently incubated with the respective HRP-linked anti-rabbit or anti-mouse IgG (1:1000) (Cell Signaling Technology, Danvers, MA, USA) antibodies and developed using ECL or ECL Plus (WesternBright Sirius Chemiluminescent Detection Kit, Advansta Inc., USA). Detection and densitometric quantification of band intensities was performed using a G-Box system (Syngene, Cambridge, UK).