Ab128854

GC cells were laid in 10 cm culture dishes and cultured to 75% to 90% confluency and harvested in lysis buffer containing proteases and phosphatase inhibitors, leave it at 4 °C for about 45 min. The protein was quantified by BCA analysis. Then the proteins were isolated on SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes, these membranes were probed using primary antibodies and secondary antibodies according to the supplier's recommendations: DPYS (1:500, Proteintech, 13,237–1-AP, Wuhan, Hubei), NT5E (1:1000, Abcam, ab133582, Suite Cambridge, USA), UPP1 (1:1000, Abcam, ab128854, Suite Cambridge, USA), β-actin (1:1000, #3700, CST, USA), anti-rabbit secondary antibody (Abcam, ab150077, Cambridge, USA) and anti-mouse secondary antibodies (Bio-Rad, 1706516, Hercules, CA). Finally, enhanced chemiluminescence (ECL) was used to observe the results.

Whole-cell lysates were extracted using radio-immunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Sigma, USA), and the proteins were separated by SDS-PAGE, followed by transfer onto nitrocellulose membranes (Millipore, Bedford, USA). The membranes were then blocked and incubated with individual detection antibodies against UPP1 (Abcam; ab128854), ENO1 (Abcam; ab155102), LDHA (Invitrogen; PA5-23036), H3K27ac (Abcam; ab45173), H3K9ac (Abcam; ab10812), and GAPDH (Proteintech; 60004-1-1G) at 4°C overnight. After washing with TBST three times for 5 min each, the membranes were probed with HRP-conjugated secondary antibody (Beyotime, Shanghai, China; A0208, A0216) at room temperature for 1 h. Finally, the membranes were visualized using an enhanced chemiluminescence system (Bio-Rad, USA). GAPDH was employed as an internal control.