Western chemiluminescent hrp substrate kit

The cells were lysed by ice-cold RIPA lysis buffer (Biyuntian, China) containing protease inhibitors, and the protein concentration was quantified using a BCA protein assay kit (Biyuntian). Each group of proteins was denatured by boiling, and then sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins. Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by electrophoresis. Then, the membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4 °C in diluted primary antibody. Finally, images of the target strip were developed using a Western Chemiluminescent HRP Substrate Kit (Millipore, USA). Image Lab software (Bio-Rad, USA) was used for semi-quantitative analysis. Primary antibodies directed against AMPK (D5A2 for monoclonal antibody, Cell Signaling Technology), p-AMPK (40H9 for monoclonal antibody, Cell Signaling Technology), β-catenin (polyclonal antibodies, Cell Signaling Technology), p-β-catenin (polyclonal antibodies, Cell Signaling Technology), p-Smad-1/5/8(D5B10 for monoclonal antibody, Cell Signaling Technology), Smad-1/5/8 (N-18 for monoclonal antibody, Santa Cruz Biotechnology) and GADPH (6C5 for monoclonal antibody, Beyotime) were used.

Total proteins were extracted from cells or tissues with TNEN buffer containing phosphatase and proteinase inhibitors, quantitated by the Bradford method (Bio‐Rad [Hercules, CA, USA] assay), and subjected to SDS‐PAGE gel electrophoresis, which were transferred onto nitrocellulose membranes. The proteins were detected with specific antibodies using standard Western blot method. The following antibodies were used: β‐Actin (Santa Cruz [Dallas, TX, USA], sc‐47778), HB‐EGF (Santa Cruz, sc‐365182), p‐Akt1 (CST, 9271S), Akt1 (CST, 9272S), p‐Erk (CST, 4377), Erk (CST, 9102S), p‐EGFR (CST, 3777s), EGFR (Abcam, ab5644), p‐Smad1/5/8 (CST, 9511l), Smad1 (CST, 9473l). Immunoreactivity was detected using a Western Chemiluminescent HRP Substrate Kit (Millipore, Burlington, MA, USA) and imaged with FluorChem M system (ProteinSimple, San Jose, CA, USA).

P. aeruginosa strains were grown overnight in LB broth at 37°C with shaking at 200 rpm. Precultures were used to inoculate (initial turbidity OD_600nm 0.05) LB supplemented with 10 mM MgCl₂, 0.5 mM CaCl₂ and 5 mM EGTA pH 7.4. Bacterial cultures were grown under the same conditions until they reached a turbidity of OD_600nm 0.8. One mL of each culture was centrifuged at 16,300 x g for 2 min. The supernatant was transferred to a 1.5 mL microtube and centrifuged again to remove residual bacteria. Nine-hundred milliliters of supernatant was taken, and 100 mL of trichloroacetic acid (TCA) was added and incubated for 12 h at 4°C. Precipitated proteins from the supernatant were harvested by centrifugation at 18,100 x g at 4°C for 30 min. The resulting protein pellet was resuspended in 1X SDS-Page sample buffer with 10% v/v saturated TRIS, and the buffer sample volume was normalized according to turbidity of each culture. The secreted proteins were solved on 15% SDS-PAGE and transferred to a nitrocellulose membrane, blocked overnight with 5% w/v non-fat milk in TBS-T (Tris buffered saline with 0.1% v/v Tween 20). The membrane was rinsed with TBS-T and probed against anti-ExoU polyclonal antibodies. The immunoblotting was carried-out using a Western Chemiluminescent HRP Substrate Kit (Millipore), and protein bands were visualized on a C-Digit Blot Scanner (Li-Cor).

BMSCs were seeded into 6-cm-diameter culture dishes and cultured in complete medium. EMD with a concentration of 5, 25, 50 μg/ml was added to the experimental group, respectively. And, the same amount of complete medium was added to the control group. The cells were collected after 3 days of culture. Total proteins were extracted from the BMSCs by ice-cold RIPA lysis buffer. Subsequently, lysate proteins were resolved by electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Then, the membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4℃ in diluted primary antibody. Finally, images of the target strip were developed in the way of using a Western Chemiluminescent HRP Substrate Kit (Millipore, USA). Primary antibodies directed against Runx2 (1:1500, Rabbit mAb#12,556, monoclonal antibody, Cell Signaling Technology, MA, USA), Osterix (1:1000, ab209484, monoclonal antibody, Abcam, UK), ALP (1:1500, ab5462, monoclonal antibody, Abcam, UK), β-catenin (1:6000, ab2572, Abcam, UK) and GAPDH (1:1000, bs-2188 R, Bioss, China). ImageJ was used to analyze the gray value of each band.

We applied 10 and 12% concentration of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein lysates and transferred them onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Then, membranes were incubated with primary antibodies specific for SCARA5 (#ab118894, Abcam), FOXM1 (#ab207298), CyclinB1 (Sc245), CHK1 (A5004, Bimake), CDC25C (A5133, Bimake), CDC25C (Ser216) (#4901, CST), CDK1 (SC54), Phospho-HistoneH2AX (Ser139) (#80312, CST), HSPA5 (#3177, CST). Protein bands were exposured with Western Chemiluminescent HRP Substrate kit (Millipore Corporation, Billerica, MA, USA).