Molecular imager chemidoc xrs gel imagine system

The detailed procedure was reported previously [26 (link), 30 (link)]. Briefly, cell lysates containing equal amounts of protein concentration were separated on 10 % SDS polyacrylamide gels. Membranes (Millipore, Shanghai, China) were incubated with antibodies against p38 MAPK, p-p38 MAPK, FOXO3a and IGFBP1 (1:1000). The membranes were washed and incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, Shanghai, China). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millpore, Shanghai, China), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (BioRad, Hercules, CA, USA) and documenting the results.

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).

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.

The Western blot procedures were performed as reported previously^{17 (link), 71 (link)}. Briefly, after determining the protein concentrations by the Bio-Rad protein assay. The cell lysates containing equal concentration of protein were solubilized in SDS-sample buffer and separated on SDS polyacrylamide gels. Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against phosphor- and total Stat3, DNMT1 and EZH2 (1:1000 and 1:2000, respectively). The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase, and transferred to freshly made ECL solution (Immobilon Western; Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (BioRad, Hercules, CA, USA) and documenting the results.

The detailed procedure was reported previously 17, 36. In brief, equal amounts of protein from cell lysates were solubilized and separated on SDS polyacrylamide gels. Membranes were incubated with antibodies against EP4, DNMT1, c‐Jun, total and phosphor‐form (Thr202/204) of ERK1/2, followed by incubating with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., Beverly, MA, USA). Afterwards, the membranes were visualized and enhanced chemiluminescence (Immobilon Western; Millipore, Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (Bio‐Rad, Hercules, CA, USA) and documenting the results.