0.22 mm pvdf membrane

Lysis buffer was used to lyse the cells, and BCA protein assay kit (Thermo Fisher Scientific) tested the concentration of the protein. Then, the protein was isolated with 10% SDS-PAGE and moved to 0.22 mm PVDF membrane (Millipore, Billerica, MA, United States) for electrophoresis. Later on, the membrane was blocked with phosphate buffer saline involving 5% bovine serum albumin for 2 h at room temperature. Afterwards, it was incubated with primary antibodies with different dilutions at 4°C overnight. The primary antibodies were IGF2BP1 (abcam, Cambridge, United Kingdom), GAPDH (abcam, Cambridge, United Kingdom), and Ki67 (abcam, Cambridge, United Kingdom). Goat anti-rabbit IgG H&L (HRP) was the secondary antibody. The protein level in each sample was normalized with respect to GAPDH. Each assay involved 3 repeated wells of each sample and all of them were repeated at least three times.

NCI-H23 and A549 cells were lysed with RIPA lysis solution (Beyotime) containing a mixture of protease inhibitors. The cell protein lysis product was separated by 10% SDS-PAGE electrophoresis, transferred onto a 0.22 mm PVDF membrane (Millipore), and detected with a specific antibody. A chemiluminescent substrate was added to the specific strip and quantified with densitometry (Quantity One software, BioRad). The GAPDH antibody was used as a control. Anti-Caspase-3 antibody, cleavage Caspase-3, poly (ADP-ribose) polymerase, cleavage PARP, BAX, BAK, cyclin D3 and CDK4 (1:1000) were purchased from Cell Signaling Technology. Anti-SIRT6 antibody was purchased from Abcam. BCL-2 and BCL-XL antibody were purchased from Proteintech.

Total protein from cells or tissues was harvested with ice-cold RIPA buffer (10 mM Tris, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1% NP-40, pH 7.4) plus protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Roche). Equivalent amounts of protein were separated by 10% or 12% SDS–PAGE and transferred onto a 0.22-mm PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% skimmed milk (w/v) in TBST buffer for 1 h and incubated with the rabbit polyclonal to AGE antibody (Abcam, 1:1000) with gentle rocking overnight at 4°C. The membranes were further incubated with goat anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling Technology, 1:1500) for 1.5 h at room temperature. Finally, protein bands were imaged using an enhanced chemiluminescence detection kit (Pierce ECL Plus, Thermo Scientific) and analyzed with a luminescent image analyzer (ImageQuant LAS 4000 or Amersham Imager 600, GE Healthcare Life Sciences).

The SCC‐1 and SCC‐47 cell lines were seeded onto 6 cm dishes with 5 × 10⁵ cells per dish and treated with different concentrations of hellebrigenin (0, 2, 4 and 8 nM) for 24 h. Next, the RIPA cell lysis buffer containing protease/phosphatase inhibitor cocktails (EMD Millipore) was added to the cells to extract protein. After quantification with BCA Cell Quantification (Thermo Fisher Scientific) assay, the protein samples were mixed with dye (1:4) and separated on 12.5% and 15% polyacrylamide gel, followed by transfer onto a 0.22 mm PVDF membrane (EMD Millipore). Afterwards, the membrane was blocked with 5% skim milk for 1 h at room temperature and incubated with the indicated primary antibodies overnight at 4°C. The next day, appropriate secondary antibodies were added to the membrane, and the protein bands were visualized with chemiluminescence fluorescence Image Quant LAS 4000 (GE Healthcare, Berlin, Germany) biomolecule imaging system.

Cells were lysed with a RIPA lysis solution (Beyotime, Shanghai, China) containing protease inhibitors. Protein lysates were separated by 10% SDS/PAGE electrophoresis, transferred onto 0.22‐mm PVDF membranes (Millipore) and incubated with specific antibodies. The β‐actin antibody (diluted 1/1000) purchased from Abcam was used as a loading control. The anti‐E‐cadherin antibody (diluted: 1/10 000), anti‐vimentin antibody (diluted: 1/2000), anti‐cyclin D1 (diluted: 1/200) and anti‐P19 (diluted: 1/1000) were purchased from Abcam. The anti‐CDK6 antibody (diluted 1/1000) was purchased from Cell Signaling Technology (Danvers, MA, USA). The anti‐MMP9 antibody (diluted 1/1000) and anti‐CCL5 antibody (diluted 1/1000) were purchased from Affinity. The anti‐mouse and anti‐rabbit IgG were purchased from Abcam and diluted to 1/2000 when applied. Autoradiograms were quantified by densitometry (quantity one software; Bio‐Rad, Hercules, CA, USA). imagej software (National Institutes of Health, NIH, Bethesda, MD, USA) was used to calculate the grey value of the western blot band to compare changes in protein levels.

Cell lysates were electrophoretically separated by 10% SDS-PAGE, transferred to 0.22-mm PVDF membranes (Millipore, USA), and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Tannon, China). GAPDH antibody was used as a control. Other antibodies were listed in Supplementary Table S2.

Cells were lysed in RIPA cell lysis buffer (Kangwei, Beijing, China) containing a phosphatase inhibitor cocktail (Sigma). Equal protein concentrations were loaded onto 10% SDS-PAGE gels and then transferred onto 0.22 mm PVDF membranes (Millipore, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C and subsequently with HRP-conjugated secondary antibodies before visualization using ECL reagent (Millipore, MA, USA).