Ps107

Cell lysis buffer for Western and IP (P0013, Beyotime) was used to lyse HGC27 and AGS cells after they had been exposed to various doses of NTP for 24 h. The BCA protein Colorimetric Assay Kit (E-BC-K318-M, Elabscience) was subsequently utilized to determine the total protein. After the protein was separated by electrophoresis, the membrane was transferred using polyvinylidene difluoride (PVDF) at an ice bath temperature. Five percent skim milk was used to block the portions. In a 4 °C shaker, the matching antibodies were incubated overnight. After rinsing with Tris-buffered saline and Tween (TBST), the film was treated with secondary antibodies (S0002, Affinity) for 1.5 h at room temperature. Finally, the imaging system was used to build the improved ECL chemiluminescent substrate kit (36222, Yeasen). For some of the PVDF membranes, Western blot fast stripping buffer (PS107, Epizyme) was used. After completing Western blot luminescence detection, the PVDF membranes were rinsed with TBST for 5 min. Add 10 mL of fast stripping buffer to cover the membrane and rinse for 20 min. The stripping solution was removed, and TBST was added to rinse the PVDF membrane for 5 min. The membrane was sealed with skim milk powder for 2 h, the PVDF membrane was washed with TBST, and then the other antibodies were added again. The gray value was analyzed and calculated using ImageJ software.

For immunoblotting, samples were lysed in 2× SDS-loading buffer and boiled for 3 min at 95 °C. Proteins were separated by SDS-PAGE on Precast 4–12% Bis-Tris gels (Biofuraw, 180-8008H) and transferred onto nitrocellulose membrane with 0.2 μm pore size (GE Healthcare, 10600001). After three washes in TBST (150 mM NaCl, 50 mM Tris-HCl, 0.05% Tween 20, pH 7.5), the membrane was incubated in blocking buffer (5% skimmed milk powder in TBST) for 1 h, followed by incubation with primary antibodies in antibody dilution buffer (4% BSA in TBST) overnight at 4 °C. After three washes in TBST for 15 min each, the membrane was incubated with HRP-conjugated secondary antibodies in antibody dilution buffer for 1 h and washed for 15 min three times. Western Lightning Plus-ECL (PerkinElmer, NEL104001EA) was used to detect proteins on membranes, and the luminescent signals were captured with X-ray films (Fig. 1c) or MiniChemi Chemiluminescence imager (Sagecreation) (Figs. 4f, 6c, 7b and Supplementary Fig. 1a). After the detection of IFT81 and RPL4 (Fig. 1c), the immunoblots were stripped off previous antibodies by incubating in stripping buffer (EpiZyme, PS107) for 15 min and used to detect eIF3d and eIF3h, respectively.
Antibodies for immunoblotting were listed in Supplementary Table 3.