Sds loading buffer

Equal amounts of each protein diluted in 5 × sodium dodecyl sulfate (SDS) loading buffer (Beyotime, Shanghai, China) were separated using SDS–polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). After being blocked at room temperature for 1 h, the membranes were incubated with primary antibodies against GAPDH (1:1000, Cell Signaling Technology, Massachusetts, USA, 5174), p21 (1:1000, Abcam, Cambridge, UK, 109,520), p53 (1:1000, Cell Signaling Technology, 48,818), p16 (1:1000, Abcam, 51,243), SERPINE2 (1:1000, Abcam, 134,905), YBX3 (1:2000, Bethyl, Montgomery, USA, A303-070A), β-Tubulin (1:1000, Cell Signaling Technology, 2128), PCNA (1:1000, Abcam, 29), Histone3 (1:1000, Abcam, 176,842), ubiquitin (1:1000, Abcam, 134,953), HNRNPC (1:1000, Abcam, 133,607), YBX1 (1:1000, Proteintech, Rosemont, USA, 20,339–1-AP), and ZO-1 (1:1000, Proteintech, 21,773–1-AP) overnight at 4 °C. The membranes were washed 3 times, incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, BOSTER, California, USA, BA1050, BA1054) for 1 h at room temperature and then washed 3 times with TBST. Specific immunoreactive bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The mean intensity ratio was analyzed by ImageJ 1.4.

RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor (Pierce, Bradenton, Florida, USA) was used to extract the total protein. The lysates were centrifuged at 1360 g for 7 min, and the supernatant was boiled in sodium dodecyl sulfate (SDS) loading buffer (Beyotime, Shanghai, China) for 10 min. After separation on 12% acrylamide SDS-PAGE gels, the protein bands were transferred onto a polyvinylidene difluoride membrane (CST, Danvers, Massachusetts, USA). The membrane was then blocked in 5% defatted milk. To expose more different proteins, we cut the whole membrane before hybridizing the antibodies, and then incubated the different primary antibodies (Abcam, Cambridge, UK. 1:1000) at 4 °C overnight. After cleaning, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK. 1:20000). Protein bands were visualized using chemiluminescence reagent (Millipore, Massachusetts, USA) and analyzed using Quantity One 4.6.3 Image software, as described previously [15 (link)].

48 h after the transfection of HeLa cells, the culture medium was removed. After PBS rinsing, the cells were lysed with protein lysate (Beyotime, China), and the total proteins were collected. Then the protein concentration was detected by BCA (Beyotime, China) protein quantitative kit. After adding proper amount of SDS loading buffer (Beyotime, China), denaturation was carried out in boiling water at 100°C for 5 min. Then 12% SDS-PAGE electrophoresis (Beyotime, China) was performed. Protein bands were transferred to PVDF membranes (Beyotime, China) by western transmembrane system. Subsequently, the PVDF membrane was sealed in 5% skim milk powder (Beyotime, China) and incubated for 4 h. After washing with TBST (Beyotime, China), the PVDF membrane was incubated with primary antibody at 4°C overnight. After washing, the membrane was incubated with the secondary antibody at room temperature for 60 min. Finally, the PVDF membrane was stained with western TMB substrate (Beyotime, China; P0211) and scanned. The antibodies used were β-actin (Beyotime, China; AF5003), TYMS (ABCAm, United States, ab108995) and horseradish peroxidase labeled goat anti-rabbit IgG (Beyotime, China; A0208).

For immunoprecipitation, equal amounts of protein from lysates prepared as above were co-incubated with Trx primary antibody and protein A magnetic beads (Santa Cruz) at 4°C overnight. The pellet was washed five times with ice-cold NP40 (Beyotime), resuspended in SDS-loading buffer (Beyotime), and analyzed by immunoblotting with Trx and ASK1 antibodies, respectively.

QSG7701 and SNU449 cells were collected and lysed in RIPA buffer (Beyotime, China) supplemented with PhosSTOP (Roche, USA) and 1 mM PMSF (Beyotime, China). Proteins were mixed with SDS loading buffer (Beyotime, China) and boiled for 5 min. Then, the proteins were loaded into 4-12% SDS PAGE gels (Genescript, China) and subjected to electrophoresis, after which the proteins were transferred to PVDF membranes and went through antibody incubation. Primary antibodies used were as follows: PRC1 (#6290001, Biolegend, USA) and GAPDH (#2118, CST, USA). GAPDH was used as a loading control.

Sorted DC subsets were lysed in cold RIPA Lysis Buffer (Beyotime Biotechnology) supplemented with protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail (Bimake), and SDS loading buffer (Beyotime Biotechnology). Total protein was denatured and loaded into the SDS-PAGE, and then transferred to PVDF membranes. After blocking, primary antibodies were hybridized overnight (HDAC3, 10255–1-AP, ProteinTech; β-actin, 3700, CST) and then secondary antibodies. Imaging was carried out with Amersham Imager 600 System (GE).