Secondary goat antibody raised against rabbit igg conjugated to horseradish peroxidase

The detailed procedure was reported previously64 (link). Briefly, protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein from whole cell lysates were solubilized in SDS-sample buffers and separated on SDS polyacrylamide gels. Membranes were incubated with antibodies against MUC1, p65, p50, the phosphor and total AMPKα. The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Beverly, MA, USA). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millpore, Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (Bio-Rad, Hercules, CA, USA).

Protein concentrations were determined by the Bio-Rad protein assay. Equal amounts of protein from whole cell lysates were solubilized in 2x SDS-sample buffer and separated on 10% SDS polyacrylamide gels. Membranes were incubated with antibodies against PDK1, PPARg phosphor-AMPKα (thr172) phosphor-p-SAPK/JNK (Thr183/Tyr185) and total AMPKα and SAPK/JNK, p53, p65 and Egr-1. The membranes were washed and incubated with incubation with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, Beverly, MA, USA). The membranes were washed again and transferred to freshly made ECL solution (Pierce, Rockford, IL, USA) for 1 min, and exposed to X-ray film.

The detailed procedure was reported previously (Zhao et al., 2015). Protein concentrations were estimated using the Bio‐Rad protein assay kit (Hercules, CA, USA). Samples were resolved by 5 × SDS‐sample buffer and separated on SDS polyacrylamide gels. Each membrane was blocked in 5% (w/v) nonfat milk in Tris‐buffered saline with 0.1% (v/v) Tween‐20 for 1 hr followed by incubating with specific primary antibodies against SP1 and β‐actin (1:1000) at 4°C overnight. The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Beverly, MA, USA) and were visualized using HRP‐conjugated anti‐rabbit secondary antibodies and enhanced chemiluminescence (Immobilon Western; Millpore, Billerica, MA, USA) followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (Bio‐Rad, Hercules, CA, USA). Image software (National Institutes of Health, Bethesda, MD, USA) was applied to quantify and compare the intensity of single band between the control and proteins of interest.