Vinculin antibody 7f9

Expression of p70S6K, phospho-p70S6K, NFκB-p65 and phospho-NFκB-p65 in primed smooth muscle cells were measured in cell lysate by western blot. Cells were primed with oxLDL as described above and lysed after 4 h. To analyze expression of Hif1α, cells were primed with oxLDL as described above and harvested on day 5. 7.5 to 10% acrylamide gels were loaded with equal amount of protein per lane. Proteins were blotted onto a PVDF membrane and blocked in 5% milk (w/v) in Tris-buffered saline supplemented with Tween 20 (TBS-T). Membranes were incubated in primary antibody [p70S6 Kinase (49D7) Rabbit Antibody, Cell Sigaling, 2708; Phospho-p70S6 Kinase (Thr389) Antibody, Invitrogen 710095; phospho-NFκB p65 (Ser536) Antibody, Cell Signaling 3033; NFκB p65 (F-6), Santa Cruz, Sc-8008; Hif-1α Antibody, BD 610959; Vinculin Antibody (7F9), Santa Cruz, Sc-73614] in TBS-T overnight at 4°C, washed 3 times and incubated in secondary antibody (Goat anti-rb IgG- HRP, Santa Cruz, sc-2004; Goat anti-mouse IgG-HRP Santa Cruz, sc-2005) in TBS-T for 1 h. For development of the membranes Pierce ECL Western Blotting Substrate (ThermoFisher, 32106) was used.

Protein expression of pro-CASP1, CASP1, pro-IL-1β and IL-1β was estimated using Western blotting. 2 × 10⁶ human monocytes were seeded in a 6 well cell culture plate. The cells were treated with 2 μM T1317 or left untreated for 24 h and lysed for western blotting. Equal amounts of proteins were loaded on 15% SDS-PAGE. Proteins were transferred on a PVDF membrane (GE Healthcare, #10600023) and blocked in 5% milk (w/v) in Tris–buffered saline supplemented with Tween 20 (TBS-T). Membranes were incubated with a primary antibody pro-CASP1 (Cell Signaling, #3866), cleaved CASP1 (Cell Signaling,#4199), pro-IL-1β (Cell Signaling,#12703); IL-1β (Cell Signaling,#83186); Vinculin Antibody (7F9) (Santa Cruz, Sc-73614), in TBS-T overnight at 4°C, followed by washing with TBST and incubation with a secondary antibody (Goat anti-rb IgG-HRP, Santa Cruz, #sc-2004 or Goat anti-mouse IgG-HRP, Santa Cruz, #sc-2005) in TBS-T for 1 h. The blots were washed as described above, developed using Pierce western blotting substrate (Thermo Scientific, #32106) and images were captured using an Amersham Imager 600 (GE Healthcare). The intensity of bands was quantified and normalized using ImageJ software.