Erα sirna sc 29305

Estradiol (E2758, purity ≥ 98%), MPP dihydrochloride hydrate (M7068, purity ≥ 97%), PHTPP (SML1355, purity ≥ 98%) and 3-Methyladenine (3-MA, M9281, purity ≥ 99%) were purchased from Sigma (Saint Louis, USA). Antibody for NLRP3 (19771-1-AP) and IL-18 (10663-1-AP) were purchased from Proteintech (Wuhan, China). Antibody for caspase-1 (48847) was purchased from SAB signalway antibody (Maryland, USA). Antibody for GSDMD (A18281) and IL-1β (A16288) were purchased from ABclonal (Wuhan, China). Antibody for Beclin 1 (ab210498), LC3B (ab192890), SQSTM1 (ab56416), Hcy (ab15154), estrogen receptor alpha (ERα) (ab32063), estrogen receptor beta (ERβ) (ab3576), GSDMD (ab219800), and β-actin (ab8226) were purchased from Abcam (Cambridge, UK). FITC - goat Anti-mouse IgG (ab6785), TRITC - goat anti-rabbit IgG (BS10250), FITC - goat anti-rabbit IgG (BS10950), HRP - goat anti-rabbit IgG (BS13278), and HRP - goat anti-mouse IgG (BS12478) were purchased from Bioworld (Bloomington, USA). The prestained protein MW marker (26617) were purchased from Thermo scientific. Control siRNA and ERα siRNA (sc-29305) were purchased from Santa Cruz (Santa Cruz, USA).

Human VSMCs were seeded in a 6-well culture plate at a density of 2×10⁵ cells in 2 ml antibiotic-free normal growth medium supplemented with 10% FBS. The cells were incubated at 37°C in a CO₂ incubator until the cells reached 60% confluence. Cells were then transfected with a transfection mixture composed of ERα siRNA (sc-29305) or control siRNA (sc-37007) and siRNA transfection reagent (sc-29528) (all Santa Cruz Biotechnology, Inc.) according to the manufacturer's protocols. After 6 h, cells were washed and cultured for 18 h in complete medium, and were treated with Ang II and/or LWDHF for 24 h. The cells were subsequently lysed and collected for western blot analyses.