Alkaline phosphatase linked secondary antibodies

Western blotting of rat cortical neurons whole-cell lysates was performed as previously described by our group [18 (link)]. Briefly, cell lysates were separated by electrophoresis in SDS-PAGE and, transferred electrophoretically to PVDF membranes (Millipore). After blotting, the membranes were blocked in 5 % non-fat milk in Tris-buffered saline containing 0.1 % Tween 20 (TBS-T) for one hour at room temperature and then incubated overnight with the primary antibodies at 4° C. The primary antibodies used were: rabbit polyclonal anti-LC3B, anti-SQSTM1, anti-phospho-AKT (Ser⁴⁷³), anti-AKT, anti-phospho-p44/42 MAPK (MEK/ERK1/2) (Thr²⁰²/Tyr²⁰⁴), anti-p44/42 MAPK (MEK/ERK1/2), anti-phospho-MTOR (Ser²⁴⁴⁸) and anti-MTOR, all at a dilution 1:1000, from Cell Signaling Technologies. Thereafter, the membranes were incubated with alkaline phosphatase-linked secondary antibodies, specific to rabbit IgG or mouse IgG in a 1:10000 dilution (GE Healthcare). Protein immunoreactivity was detected by chemifluorescence using the ECF substrate (GE Healthcare) in a VersaDoc Imaging System (Bio-Rad) and protein bands optical density was quantified with the Quantity One Software (Bio-Rad). Membranes were reprobed for α-Actin (Sigma; 1:10000 dilution) for equal protein loading control.

Lipopolysaccharide (LPS) from Escherichia coli (serotype 026:B6) was purchased at Sigma Chemical Co. (St. Louis, MO, United States). Fetal bovine serum (FBS) and trypsin were obtained from Invitrogen (Paisley, OR, United Kingdom). The protease and phosphatase inhibitor cocktails were obtained from Roche (Mannheim, Germany). Antibodies against phospho-IκB-α and IκB-α, were purchased from Cell Signaling Technologies (Danvers, MA, United States); against iNOS were from R&D Systems (Minneapolis, MN, United States) and against COX-1 and COX-2 from Abcam (Cambridge, United Kingdom). The anti-actin antibody was purchased from Millipore (Bedford, MA). The alkaline phosphatase-linked secondary antibodies and the enhanced chemifluorescence (ECF) reagent were obtained from GE Healthcare (Chalfont St. Giles, United Kingdom). The polyvinylidene difluoride (PVDF) membranes were from Millipore Corporation (Bedford, MA). Trizol^® reagent was purchased from Invitrogen (Barcelona, Spain). iScript kit and SYBR green were obtained from BioRad (Hercules, CA, United States). Primers were from MWG Biotech (Ebersberg, Germany). The remaining reagents were from Sigma Chemical Co. (St. Louis, Mo, United States) or from Merck (Darmstadt, Germany).