Western blotting assay was prepared as previously described [14 (link)]. A 10% polyacrylamide gel and Bio-Rad mini-Protean III apparatus (Bio-Rad) were used to separate the membrane-enriched proteins (15 µg) by electrophoresis (SDS-PAGE; 90 V). The proteins were blotted onto a nitrocellulose membrane (Bio-Rad) and incubated overnight with the specific primary antibodies (1:1000) TNFR2 (sc-7862, Santa Cruz Biotechnology), NR2A, pAMPA, AMPA, and NR1 (4205, 8084, 13,185, and 5704, Cell Signaling Technology). The membranes were then incubated for 2 h with a specific secondary antibody.
Proteins recognized by antibodies were detected using electrochemiluminescence (ECL). To standardize and quantify the immunoblots, we used ImageJ software (NIH). The β-ACTIN antibody (sc-1616; Santa Cruz Biotechnology) was used as an internal experimental control, with results expressed concerning β-ACTIN intensity.
Proteins recognized by antibodies were detected using electrochemiluminescence (ECL). To standardize and quantify the immunoblots, we used ImageJ software (NIH). The β-ACTIN antibody (sc-1616; Santa Cruz Biotechnology) was used as an internal experimental control, with results expressed concerning β-ACTIN intensity.
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