Horseradish peroxidase linked anti mouse or rabbit igg antibodies

After agmatine treatment and exposure to high glucose stress, N2A cells were washed twice with ice-cold PBS, scraped, and collected. N2A cell pellets were lysed with ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). The lysates were centrifuged at 16,100g for 1 hour at 4℃ to produce whole-cell extracts. Protein content was quantified using the BCA protein assay kit (Pierce, IL, USA). Protein (35 µg) was separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% bovine serum albumin in TBS/Tween (20 nM Tris (pH 7.2), 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature, immunoblots were incubated overnight at 4℃ with primary antibodies that specifically detect cleaved caspase 3 (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 (1:2000, Abcam, Cambridge, MA, USA), phosphor-p53 (1:2000, Abcam, Cambridge, MA, USA), p53 (1:2000, Abcam, Cambridge, MA, USA), or β-actin (1:2000, Cell Signaling, Danvers, MA, USA). Blots were then incubated with horseradish peroxidase-linked anti-mouse or -rabbit IgG antibodies (Abcam, Cambridge, MA, USA) for 2 hours at room temperature. Protein bands were detected and analyzed using enhanced chemiluminescence (ECL; Pierce, IL, USA).

After treatment with LPS and miR-Let7A transfection, cells were washed rapidly with ice-cold PBS, scraped, and collected. THP-1 cell pellets were lysed with ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA), and the lysates were centrifuged at 13,000 rpm for 1 hr 30 min at 4℃ to produce whole-cell extracts. Protein content was quantified using the BCA method (Pierce, Rockford, IL, USA). Protein (35 µg) was separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% BSA prepared in TBS/Tween (20 nM Tris (pH 7.2), 150 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature, immunoblots were incubated overnight at 4℃ with primary antibodies specifically for detection of caspase-3 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 (1:1000, Santa Cruz Biotechnology), ASK1 (1:1000; Santa Cruz Biotechnology), phosphorylated ASK1 (p-ASK1; 1:1000; Santa Cruz Biotechnology), or β-actin (1:1000; Cell Signaling, Danvers, MA, USA). Blots were then incubated with horseradish peroxidase-linked anti-mouse or -rabbit IgG antibodies (Abcam, Cambridge, MA, USA) for 1 hr at room temperature. Enhanced chemiluminescence was performed by ECL (Pierce).